AU2015219289B2 - Methods and compositions for DNA profiling - Google Patents

Abstract

Embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a SNP and at least one target sequence comprising a tandem repeat, and determining the genotypes of the at least one SNP and at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample. Embodiments disclosed herein further provide a plurality of primers that specifically hybridize to at least one short target sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single reaction results in a short amplification product and a long amplification product, wherein each of the plurality of primers comprises one or more tag sequences.

Description

METHODS AND COMPOSITIONS FOR DNA PROFILING

RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional Application No. 62/103,524 filed January 14, 2015, U.S. Provisional Application No. 62/043,060 filed August 28, 2014, and U.S. Provisional Application No. 61/940,942 filed February 18, 2014, the contents of which are incorporated herein by reference in their entireties.

REFERENCE TO SEQUENCE LISTING, TABLE, OR COMPUTER PROGRAM LISTING

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled ILLINC276WO Sequence Listing.TXT, created February 13, 2015, which is 55 Kb in size. The information in the electronic format of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD OF THE DISCLOSURE

[0003] Embodiments provided herein relate to methods and compositions for DNA profiling. Some embodiments relate to methods of amplification of target sequences of variant sizes in a. single reaction, followed by subsequent sequencing of the library.

BACKGROUND OF THE DISCLOSURE

[0004] Historically, the use of a. subset of markers in a human genome has been utilized to determine an individual's personal identity, or DNA fingerprint or profile. These markers include locations or loci of short tandem repeated sequences (STRs) and intermediate tandem repeated sequences (ITRs) which in combination are useful in identifying one individual from another on a genetic level. The analysis of these markers has become standardized in the analysis of DNA found at crime scenes. For example, in the United States a. number of these repeated sequences have been combined to create a Combined DNA Index System (CODIS), which serve as the laboratory standard for DNA profiling in criminal cases. Other countries similarly have adopted a standard system for DNA profiling. These systems have also been utilized to determine paternity and familial relationships. However, the current systems are all based on size separation of these repeated loci on an electrophoretic system and are thus limited to the number of loci that can be differentiated in such a system. For example, some of the current commercial systems for DNA profiling for forensics purposes differentiate only 16 markers due to the limitations of the electrophoretic detection methods.

SUMMARY OF THE DISCLOSURE

[0005] Embodiments relate to systems and methods that are not content limited and that bring different pieces of genetic information about an individual together to provide a comprehensive, more complete DNA profile of an individual. The present disclosure describes methods and compositions that enable this profile of an individual, thereby advancing the fields of personal and forensic genomics.

[0006] DNA profiling currently uses selected biological markers for determining the identity of a DNA sample. For example, the most common analysis for determining a DNA profile is to determine the profile for a number of short tandem repeated (STRs) sequences found in an organism's genome. The analysis consists of amplifying defined STR sequences that can be up to 400 bp long that can be differentiated by size on an electrophoretic gel or by using capillary electrophoresis (CE). Electrophoresis is used to detect size changes due to differences in the number of repeated STRs at a given locus and as such the length of the PGR amplicons, which for the CE system is between 5G-500bp. To help overcome the limits imposed by size differentiation methodologies (i.e., STRs of overlapping amplicon size cannot be differentiated), current methods of DNA profiling utilize different sets of labelled primers such that amplicons that overlap in size can be labelled with different fluorescent dyes whereon, upon excitation, the emission spectra differ thereby allowing for overlapping amplicons to be differentiated using differences in the dye excitation and emission spectra. Using differentiated labeling, current, methods allow for the multiplexing of 24 different STR loci using 6 differently detectable dyes in one DNA profiling run. [0007] There are many limitations to the current DNA profiling methodologies. As previously mentioned, size differentiated systems limit the number of loci that can be discretely determined at a given time. Another limitation of the established methods for DNA profiling is that the DNA to be analyzed oftentimes is degraded and the size range of some of the markers does not accommodate degraded DMA, for example the amplicons can be larger than the size of the fragments of the degraded DNA. For degraded DNA, amplicons of 400bp are considered very long and can result in loss of amplification of those longer loci . When DNA analysts amplify degraded DNA samples to identify their STR profile, for example a sample found at a crime scene, oftentimes they are unable to detect all the loci resulting in a partial profile which can make matching a suspect in a crime scene to a crime sample difficult or impossible. As a default, with such samples, a DNA analyst has little choice and if any sample is left over, additional assays need to be performed to identify other markers which might give a clue as to the identify of the individual, such as single nucleotide polymorphisms (SNPs), mini-STRs, or mitochondrial DNA (mtDNA) analysis. However, precious sample must be expended on each assay with no certainty of success in finally identifying an individual. FIG. 1 A demonstrates the potential different paths to DNA identification, all of which are separate workflows and require aliquots of precious samples. When one or more simple workflows need to be combined and potentially repeated multiple times then the resulting process is no longer simple or an effective use of a precious sample,

[0008] Embodiments described in the present, application provide methods, compositions and systems for determining the DNA profile of an individual or organism by next generation sequencing (NGS) thereby providing a solution to the problems and limitations of current methodologies for DNA profiling. FIG. IB shows an exemplary workflow of the disclosed methods in one embodiment. Disclosed herein are methods and compositions for combining a multitude of forensically relevant markers into one assay including, but not limited to, short tandem repeats (STRs), intermediate tandem repeats (ITRs), identity informative single nucleotide polymorphisms (iSNPs), ancestry informative single nucleotide polymorphisms (aSNPs) and phenotypic informative single nucleotide polymorphisms (pSNPs). [0009] The present disclosure describes assays that overcome the limitations of current methodologies for DNA profiling. Disclosed embodiments provide methods and compositions for multiplex amplification, library preparation and sequencing of combined STRs, STRs, iSNPs, aSNPs, and pSNPs from one nucleic acid sample in a single multiplex reaction. Disclosed methods analyze a plurality of markers in one experimental assay with minimal sample handling, using low amounts of sample DNA including degraded DNA. Some embodiments described can be utilized for databanking DNA profiles and'Or DNA profiles that can be used for criminal casework. Some embodiments provide PGR methods and compositions developed to be sensitive enough to detect sub-nanogram amounts of DNA. Further, the unconventional primer design parameters allow for highly multiplexed PCR for the identification of STRs, ITRs and SNPs in one multiplex reaction. For criminal casework, the present methods and compositions incorporate unique molecule identifiers (UMls) which aide in removal of, for example, PCR and sequencing errors, stutter and the like from sequencing results. See Kivioja et al., Nat. Meth. 9, 72-74 (2012). As well, the results from, the methods and compositions disclosed herein are compatible with existing databases.

[0010] Therefore, embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat in a multiplex reaction to generate amplification products, and determining the genotypes of the at least one SNP and the at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample.

[0011 ] In some embodiments, the methods comprise generating a nucleic acid library from the amplification products. In some embodiments, the methods comprise determining the sequences of the nucleic acid library. In some embodiments, the nucleic acid sample is from a human. In some embodiments, the nucleic acid sample is from an environmental sample, a plant, a non-human animal, a bacterium, archaea, a fungus, or a virus. In some embodiments, the DNA profile is used for one or more of disease diagnostics or prognosis, cancer biomarker identification, genetic anomaly identification or genetic diversity analysis. In some embodiments, the DNA profile is used for one or more of databanking, forensics, criminal case work, paternity or persona] identification. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample. In some embodiments, each of the plurality of primers has a low melting temperature and/or has a length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a melting temperature that is less than 60 degrees C. In some embodiments, each of the plurality of primers has a melting temperature that is about 50 degrees C to about 60 degrees C. In some embodiments, each of the plurality of primers has a. length of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a length of about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a homopolymer nucleotide sequence. In some embodiments, the nucleic acid sample is amplified by polymerase chain reaction (PGR). In some embodiments, the nucleic acid sample is amplified in an amplification buffer having a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt, comprises KG, LiCl, NaCI, or a combination thereof. In some embodiments, the salt comprises KG. In some embodiments, the concentration of KG in the amplification buffer is about 100 mM to about 200 mM. In some embodiments, the concentration of KG in the amplification buffer is less than about 150 mM. In some embodiments, the concentration of KG in the amplification buffer is about, 145 mM. In some embodiments, the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof. In some embodiments, the plurality of primers specifically hybridize to at least 30 SNPs. In some embodiments, the plurality of primers specifically hybridize to at least 50 SNPs. In some embodiments, the tandem repeat is a short, tandem repeats (STR), an intermediate tandem repeat (ITR), or a variant thereof. In some embodiments, the pluralit of primers specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers specifically hybridize to at least 60 tandem repeat, sequences. In some embodiments, the nucleic acid sample comprises about, 100 pg to about 100 ng DNA. In some embodiments, the nucleic acid sample comprises about 10 pg to about 500 pg DNA. In some embodiments, the nucleic acid sample comprises about 5 pg to about 10 pg DNA, In some embodiments, the nucleic acid sample comprises genomic DNA. In some embodiments, the genomic DNA is from a forensic sample. In some embodiments, the genomic DNA comprises degraded DNA. In some embodiments, at least 50% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 80% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 90% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, at least 95% of the genotypes of the at least one SNP and at least one tandem repeat are determined. In some embodiments, each of the plurality of primers comprises one or more tag sequences. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof. In some embodiments, the one or more tag sequences comprise a primer tag. In some embodiments, the one or more tag sequences comprise a unique molecular identifier tag.

[0012] Embodiments disclosed herein provide methods of constructing a nucleic acid library comprising: providing a nucleic acid sample, and amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a. tandem repeat sequence in a multiplex reaction to generate amplification products.

[Ό013] In some embodiments, the nucleic acid sample is not fragmented prior to the amplification. In some embodiments, the target sequences are not, enriched prior to the amplification. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample. In some embodiments, each of the plurality of primers comprises one or more tag sequences. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, or a unique molecular identifier tag, or a combination thereof. In some embodiments, the methods include amplifying the amplification products with a second plurality of primers. In some embodiments, each of the second plurality of primers comprises a portion corresponding to the primer tag of the plurality of primers and one or more tag sequences. In some embodiments, the one or more tag sequences of the second plurality of primers comprise a capture tag, or a sequencing tag, or a combination thereof. In some embodiments, the methods include adding single stranded-binding protein (SSB) to the amplification products. In some embodiments, the nucleic acid sample and/or the amplification products are amplified by polymerase chain reaction (PCR). In some embodiments, the nucleic acid sample and/or the amplification products are amplified in an amplification buffer having a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt comprises KC1, LiCl, NaCl, or a combination thereof. In some embodiments, the salt comprises KC1. In some embodiments, the concentration of KCI in the amplification buffer is about 500 mM to about 200 mM. In some embodiments, the concentration of KCI in the amplification buffer is less than about 150 mM. In some embodiments, the concentration of KCI in the amplification buffer is about 145 mM.

[0014] Embodiments disclosed herein provide a nucleic acid library comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise at least one tandem repeat sequence flanked by a first pair of tag sequences and at least one single nucleotide polymorphism (SNP) sequence flanked by a second pair of tag sequences. Further provided is a nucleic acid library constructed using the methods and compositions disclosed herein. In some embodiments, the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the plurality of nucleic acid molecules.

[Ό015] Embodiments disclosed herein provide a plurality of primers that specifically hybridize to at least one short target, sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single multiplex reaction results in at least one short amplification product and at least one long amplification product, wherein each of the plurality of primers comprises one or more tag sequences.

[0016] In some embodiments, the short target sequence comprises a single nucleotide polymorphism (SNP) and the long target sequence comprises a tandem repeat. In some embodiments, the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a. combination thereof. In some embodiments, each of the plurality of primers has a low melting temperature and/or has a le gth of at least 24 nucleotides. In some embodiments, each of the plurality of primers has a melting temperature that is less than 60 degrees C. In some embodiments, each of the plurality of primers has a melting temperature that is about 50 degrees C to about 60 degrees CI In some embodiments, each of the plurality of primers has a length of at least 24 nucleotides. In some embodiments, each of the pluraiity of primers has a. length of about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a homopolymer nucleotide sequence. In some embodiments, the nucleic acid sample is amplified by polymerase chain reaction (PGR). In some embodiments, the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof. In some embodiments, the plurality of primers specifically hybridize to at least 30 SNPs. In some embodiments, the plurality of primers specifically hybridize to at least 50 SNPs. In some embodiments, the tandem repeat is a short tandem repeats (STR), an intermediate tandem repeat (1TR), or a variant thereof. In some embodiments, the plurality of primers specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers specifically hybridize to at least 60 tandem repeat sequences.

[0017] Embodiments disclosed herein provide kits comprising at least one container means, wherein the at least one container means comprises a plurality of primers disclosed herein.

[0018] In some embodiments, the kits include a reagent for an amplification reaction. In some embodiments, the reagent is an amplification buffer for polymerase chain reaction (PGR). In some embodiments, the amplification buffer comprises a salt, concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, the salt comprises KG, LiCl, NaCl, or a combination thereof. In some embodiments, the salt comprises KG. In some embodiments, the concentration of KG in the amplification buffer is about 100 mM to about 200 mM. in some embodiments, the concentration of KG in the amplification buffer is less than about, 150 mM. In some embodiments, the concentration of KG in the amplification buffer is about 145 mM. BRIEF DESCR IPTION OF THE DRAWINGS

[0019] FIG. 1A and FIG. IB show the differences in the A) current workflow for DNA profiling versus B) that of one exemplary embodiment of the present, disclosure.

[0020] FIG. 2 shows one exemplary embodiment of a method for creating a library useful for DNA profiling.

[0021] FIG 3 shows another exemplary embodiment of a method for creating a library useful for DNA profiling.

[0022] FIG. 4A, FIG. 4B, FIG. 4C and FIG. 4D are line graphs illustrating electropherogram results of how a primer pair designed by traditional methods and following established PCR primer design protocols and limitations can cause non-specific amplification of genomic targets and obscuration of desired ampiicons detection when combined with primers designed following methods of the present disclosure; A) 10 primer pairs designed by methods of the present disclosure directed to SNP loci, B) and D) the 10 primers plus an additional primer pair designed by traditional methods demonstrating that the additional primer pair interferes with the 10 primer pairs during amplification, and C) the 1 0 primer pairs plus an additional primer pair, wherein the additional primer pair was also designed by following the methods of the present disclosure resulting in a successful amplification of all the targeted SNPs. The X-axis is the size of the library fragments (bp) and the Y axis is fluorescence units of (FU) of the amplified peaks of the amplified fragments.

[0023] FIG. 5A, FIG. 5B, FIG. 5C, FIG. 5D and FIG. 5E are box plots showing exemplary results for an experiment following the workflow outlined in FIG. 2, which was used to identify a panel of 56 STRs and mix of 75 identity-informative SNPs (iSNPs), ancestry-informative SNPs (aSNPs) and phenotypic-informative SNPs (pSNPs) in a multiplex amplification and sequencing reaction from a sample. Reported are replicated results demonstrating successful amplification and sequencing of the STR loci from the panel; A) box plot demonstrating intra-locus balance for 25 heterozygous STRs from the panel, B) box plot demonstrating low stutter for the majority of the 56 STR loci, C) box plot demonstrating sequencing coverage for the STR loci, D) box plot demonstrating the sequence coverage for the SNPs, and E) box plot demonstrating balance for 22 heterozygous SNPs from the panel. The lower error bar indicates the minimum value, the upper error bar indicates the maximum value, the lower box reports the 25 ⁿ percentile and the upper box reports the 75^th percentile with the mean being the intersection between the lower and upper boxes.

[ 0024] FIG. 6 shows a series of bar charts showing the exemplary STR loci plots from, the experiment of FIG. 5. The plots show different allelic calls for the STRs in the panel of FIG. 5.

[0025] FIG. 7A, FIG. 7B, FIG. 7C, FIG. 7D and FIG. 7E are box plots showing exemplar}' results for an experiment following the workflow outlined in FIG. 3, which was used to identify a panel of 26 STRs and a. mix of 94 iSNPs, aSNPs and pSNPs in a multiplex amplification and sequencing reaction from a sample. Reported are replicated results demonstrating successful amplification and sequencing of the STRs from the panel; A) box plot demonstrating intra-locus balance for 21 heterozygous STR loci from the panel, B) box plot demonstrating low stutter for the 26 STR loci (39 of the 47 alleles of the 26 loci showed no stutter), C) box plot demonstrating sequencing coverage for the STR loci (read numbers normalized using the UMIs), D) box plot demonstrating the sequence coverage for the SNPs and E) box plot demonstrating balance for 21 heterozygous iSNPs from the panel. The lower error bar indicates the minimum value, the upper error bar indicates the maximum value, the lower box reports the 25^th percentile and the upper box reports the 75^to percentile with the mean being the intersection between the lower and upper boxes.

[0026] FIG. 8 shows a series of bar charts showing the exemplary STR loci plots from the experiment of FIG. 7. The plots show different allelic calls for the STRs in the panel of FIG. 7.

[0027] FIG. 9 shows bar graphs of samples analyzed without UMIs, and with UMIs. The left panel for each set represents samples analyzed without UMIs and the right panel for each set represents samples analyzed with UMIs. The X axis designates the repeat number of the STR and the Y axis designates the count number of the particular allele. The error lines within the bars separate the sequencing error (upper part of the bar) from the correct sequence (low er part of the bar) within the STR sequence.

[0028] FIG. 10A and FIG. 10B show exemplary results from an experiment where the DNA ratio was 90: 10 female :male. A) a subset of STR loci call results for STR loci when using current capillar}' electrophoresis DNA profiling methods, and B) several STR loci call results for several STR loci when using the methods of the present application. Both the CE methods and the methods of the present application did detect the low level of male DNA contamination.

[0029] FIG. shows bar charts that show that STR. loci specific to the Y chromosome were detected in the experiment of FIG. 9, further demonstrating that the present application can detect contaminating male DNA and specific STR loci from that male DNA whereas two experiments would need to be run with the current CE methodologies to do so.

[0030] FIG. 12 is a table that shows exemplary high level sequencing results from an experiment using 12 sample individuals and a refere ce individual, demonstrating consistency of STR and SNP calls between two replications.

[0031] FIG. 13 is a table that shows exemplary population statistics from the experiment shown in FIG. 12.

[0032] FIG. 14 is a table that shows exemplar}' phenotype predictions based on genotype of pSNPs from the experiment shown in FIG. 12.

[0033] FIG. 15 is a graph showing exemplary ancestry mapping based on genotype of aSNPs from the experiment shown in FIG. 12.

[0034] FIG. I6A, FIG. 16B, FIG. 16C, FIG. 16D and FIG. 16E are bar charts showing the exemplary STR loci plots from the experiment of FIG. 12.

[0035] FIG. 17A and FIG, 17B are bar charts showing exemplar}' SNP plots from the experiment of FIG, 12,

[0036] FIG , 18A and FIG.18B show box plots showing the intra-locus balance of exemplar}' STR and SNP loci from the experiment of FIG. 12.

[0037] FIG. 19A and FIG, 19B are graphs showing stutter analysis of exemplaiy STR loci from the experiment of FIG. 12.

[0038] FIG. 20 is a table that shows exemplary isometric heterozygotes in STR loci from the experiment of FIG. 12.

[0039] FIG. 25 is a block diagram that shows an exemplary inheritance plot based on variants with in the STR D8S 1 179 from the experiment of FIG. 12. [0040] FIG. 22 is a block diagram that shows an exemplary inheritance plot based on variants with in the STR D13S317 from the experiment of FIG. 12.

[0041] FIG. 23 is a table that shows exemplary genotyping results using degraded

DNA.

[0042] FIG. 24A and FIG. 24B show exemplary STR genotyping results and intro-locus balance at difference DNA inputs.

[0043] FIG. 25A and FIG. 25B show exemplary SNP genotyping results and intro-locus balance at difference DNA inputs.

DETAILED DESCRIPTION

Definitions

[0044] All patents, applications, published applications and other publications referred to herein are incorporated by reference to the referenced material and in their entireties. If a term or phrase is used herein in a way that is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the use herein prevails over the definition that is incorporated herein by reference,

[0045] As used herein, the singular forms "a", "an", and "the" include plural references unless indicated otherwise, expressly or by context. For example, "a" dimer includes one or more dimers, unless indicated othenvise, expressly or by context.

[0046] As used herein, the terms "DNA profile," "genetic fingerprint," and "genotypic profile" are used interchangeably herein to refer to the allelic variations in a collection of polymorphic loci, such as a tandem repeat, a single nucleotide polymorphism (SNP), etc. A DNA profile is useful in forensics for identifying an individual based on a nucleic acid sample. DNA profile as used herein may also be used for other applications, such as diagnosis and prognosis of diseases including cancer, cancer biomarker identification, inheritance analysis, genetic diversity analysis, genetic anomaly identification, databanking, forensics, criminal case work, paternity, personal identification, etc.

[0047] The terms "polynucleotide," "oligonucleotide," "nucleic acid" and "nucleic acid molecule" are used interchangeably herein to refer to a polymeric form of nucleotides of any length, and may comprise ribonucleotides, deoxyribonucleotides, analogs thereof, or mixtures thereof. This term refers only to the primary structure of the molecule. Thus, the term includes triple-, double- and single-stranded deoxyribonucleic acid ("DNA"), as well as triple-, double- and single-stranded ribonucleic acid ("RNA").

[0048] As used herein, "sequence identity" or "identity" or "homology" in the context of two nucleotide sequences includes reference to the residues in the two sequences which are the same when aligned for maximum correspondence over a specified comparison window. The portion of the nucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.

[0049] As used herein, "substantially complementary or substantially matched" means that two nucleic acid sequences have at least 90% sequence identity. Preferably, the two nucleic acid sequences have at least 95%, 96%, 97%, 98%, 99% or 100% of sequence identity. Alternatively, "substantially complementary or substantially matched" means that two nucleic acid sequences can hybridize under high stringency condition(s).

[0050] It is understood that aspects and embodiments of the invention described herein include "consisting" and/or "consisting essentially of aspects and embodiments.

[0051] Other objects, advantages and features of the present invention will become apparent from the following specification taken in conjunction with the accompanying drawings.

Methods for Constructing a DNA Profile

[0052] Established methodologies for determining the DNA profile are limited in a number of ways. For example, current methods detect size changes of amplified loci which differ due to changes in lengths of tandem repeated sequences found in a DNA sample. To multiplex STR amplifications for visualization, the amplifications have to be designed to space the different amplicons sizes within the size separation limits of the electrophoretic system, which for CE is from about 50-500bp. As such, only a limited number of the repeated sequences can be visualized in one assay. For example, the GLOBALFILER PCR amplification kit (APPLIED BIOSYSTEMS) is reportedly able to differentiate 24 STR loci by using 6 different dyes. Further, such methods have issues when sample DNA is degraded as is common with DNA samples from a crime scene, such that longer amplification products are not possible resulting in an incomplete DNA profile. Current methods are also oftentimes not sensitive enough to detect small amounts of contaminating DNA so a mixed sample can go undetected and unreported, which could be critical for criminal casework. As such, current methods can lead to incomplete results which lead to inconclusive results, which can be detrimental for DNA profiling.

[0053] Additionally, current targets do not include information about sample ancestry, phenotypic traits such as possible eye color and other individualized sample information. Some sequencing methodologies have attempted to include both STR and SNP detection. For example, library preparation followed by custom enrichment for STRs and SNPs has been attempted, however not ail STRs are completely covered as library preparation methods typically involve sample shearing that can obliterate the targeted sequence. Further, established primer design methods and protocols can provide primer sets for amplifying long sequences (e.g., STRs) or short sequences (e.g., SNPs), but the combinations of both in one reaction have not met with success.

[0054] The present disclosure describes solutions to the problems and limitations of the current DNA profiling systems. Methods and compositions described herein allow for the combination of STRs and SNPs into one assay using PCR to amplify the targets and generate libraries for sequencing. While developing the present assays, it was unexpectedly discovered that, for example, when utilizing unconventional and counterintuitive primer design, both STRs and SNPs can be amplified in one reaction which allows the sequence for all targeted loci to be determined. Surprisingly, when designing amplification primers using parameters contrary to the current dogma surrounding primer design, primers were created that allowed for the longer STR regions to be amplified and the short SNP regions to be amplified in a more or less balanced manner thereby allowing for both STRs and SNPs to be multiplex amplified together.

[0055] The methods and compositions disclosed herein for determining the DNA profile of an organism can be used whenever differently sized sets of amplicons are desired from, one amplification reaction outside of DNA profiling. For example, if targets of interest for PGR include both large gene regions and short SNP regions which may result in amplicons that vary in size from hundreds to thousands of base pairs versus amplicons of less than 100 base pairs, respectively, then the methods and compositions described herein could allow for successful simultaneous amplification of the gene and SNP targets which would not, have been possible without practicing the disclosed methods. Further, the methods and compositions disclosed herein may apply to any organism, for example humans, non-human primates, animals, plants, viruses, bacteria, fungi and the like. As such, the present methods and compositions are not, only useful for DNA profiling (e.g., forensics, paternity, individual identification, etc.) and humans as a target genome, but could also be used for other targets such as cancer and disease markers, genetic anomaly markers and/or when the target genome is not human based.

[0056] Therefore, embodiments disclosed herein provide methods for constructing a DNA profile comprising: providing a nucleic acid sample, amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat, and determining the genotypes of the at least one SNP and at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample.

[0057] It would be appreciated by those skilled in the art that any suitable techniques may be used in determining the genotypes of the target sequences including, but not limited to, array-based hybridization, sequencing, or the like. Therefore, in some embodiments, the methods disclosed herein may comprise generating a nucleic acid library, such as a sequencing library, from the amplification products, and determining the sequences of the nucleic acid library. [0058] In some embodiments, the present, disclosure provides methods and compositions for DNA profiling that comprise the concurrent identification of STRs and iS Ps, for example for use in population or personal databanking. In such databanks, personal data is not necessarily needed as the individuals are typically known. However, if additional information is desired then additional informatio targets can be added for concurrent identification. Short tandem repeats are well known in the art, and consist of repeated di- or tri nucleotide sequences. Intermediate tandem repeats are typically considered repeated sequences of between 4 to 7 nucleotide sequences. SNPs utilized herein can be of any form that might offer insight into a person's physical characteristics. Those exemplified herein are SNPs that provide clues for ancestry or heritage (aSNPs) and those that provide clues for phenotypic characteristics (phenotypic-informative SNPs). In methods described herein, a DNA profile assay might include any number of these SNPs in combination with STR and ITR loci determinations.

[0059] For example, the present disclosure provides additional methods and compositions where, along with STRs and iSNPS, additional targets are included. If more information about an individual is desired, for example when a sample belongs to an unknown individual or group of individuals as can be the case for criminal casework, the other information markers can he added to the STR and iSNPs, such as SNPs related to ancestry (aSNPs) and SNPs related to phenotypic variants (phenotypic-informative SNPs). The additional information can then be used to aid investigators, for example, by providing insight into an unknown individual's heritage, eye color, hair color, and the like. As such, the addition of all the combined information can provide a more complete DNA profile of an individual that was not previously known using current methods of DNA profiling.

[0060] The methods and compositions disclosed herein are designed to be sensitive enough to detect sub-nanogram amounts of nucleic acid molecules. Further, the methods and compositions disclosed herein may be useful to amplify a nucleic acid sample made having low-quality nucleic acid molecules, such as degraded and/or fragmented genomic DNA from a forensic sample. The nucleic acid sample may be a purified sample or a crude DNA containing lysate, for example derived from a buccal swap, paper, fabric or other substrate that may be impregnated with saliva, blood, or other bodily fluids. As such. in some embodiments, the nucleic acid sample may comprise low amounts of, or fragmented portions of DNA, such as genomic DNA. For example, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is, is about, or is less than, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 1 1 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, or is in a range defined by any two of these values, for example, 10 pg to 100 pg, 10 pg to 1 ng, 100 pg to 1 ng, 1 ng to 10 ng, 10 ng to 100 ng, etc. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is about 100 pg to about 1 ng. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is more than about 62.5 pg. In some embodiments, additional fragmentation steps, such as sonication or endonuclease digestion, are not included in the fragmentation procedures.

[0061] In some embodiments, the methods and compositions disclosed herein are capable of successfully determining the genotypes of one or more of the target, sequences, for example, SNPs, STRs, etc., even with sub-nanogram amounts of and/or degraded nucleic acid samples. For example, the methods and compositions disclosed herein are capable of successfully determining the genotype that is, is about, or is more than, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%), 90%, 95%, 96%, 97%, 98%, 99%, 100%, or a range between any two of the above values, of the target sequences. In some embodiments, the methods and compositions disclosed herein are capable of successfully determining the genotype of more than about 50%, 80%, 90%, 95%, 98% or more of the target sequences. In some embodiments, the methods and compositions disclosed herein are capable of achieve an intra- locus balance of more than about 50%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%), 98%, 99%), 100%, or a. range between any two of the above values, of the target sequences,

[0062] For forensic investigation, the plurality of primers may incorporate unique molecule identifiers (UMIs) which aide in removal of, for example, PGR and sequencing errors, stutter and the like from sequencing results. See Kivioja et al., supra. As discussed in further detail elsewhere in this disclosure, inclusion of UMI in the primers also allows the identification of variants within tandem repeat loci, further enhancing the usefulness of the curre t methods and compositions for DNA profiling and other purposes such as inherence analysis.

[0063] Accordingly, in some embodiments, the genotypes of the tandem repeat sequences as disclosed herein may include sequence variants within the tandem repeat loci. Therefore, a homozygote for a tandem repeat (e.g., 13, 13 for D9S1 122) using the traditional method may be identified as an isometric heterozygote based on sequence variants within the tandem repeat. As would be appreciated by those skilled in the art, taking into account the intra-locus sequence variants would greatly enhance the usefulness of the methods disclosed herein, for example, for inheritance analysis.

Methods for Constructing a Nucleic Acid Library

[0064] Embodiments disclosed herein provide methods of constructing a nucleic acid library comprising: providing a nucleic acid sample, and amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat sequence.

[0065] The methods and compositions disclosed herein are designed to be sensitive enough to detect sub-nanogram amounts of nucleic acid molecules. Further, the methods and compositions disclosed herein may be useful to amplify a nucleic acid sample that consists of low-quality nucleic acid molecules, such as degraded and/or fragmented genomic DNA from a forensic sample. The nucleic acid sample may be either purified or a crude DNA containing lysate, for example derived from a buccal swap, paper, fabric or other substrate that may be impregnated with saliva, blood, or other bodily fluids. As such, in some embodiments, the nucleic acid sample may comprise low amount of or fragmented DNA, such as genomic DNA. For example, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is, is about, or is less than, 1 pg, 2 pg, 3 pg, 4 pg, 5 pg, 6 pg, 7 pg, 8 pg, 9 pg, 10 pg, 1 1 pg, 12 pg, 13 pg, 14 pg, 15 pg, 16 pg, 17 pg, 18 pg, 19 pg, 20 pg, 30 pg, 40 pg, 50 pg, 60 pg, 70 pg, 80 pg, 90 pg, 100 pg, 200 pg, 300 pg, 400 pg, 500 pg, 600 pg, 700 pg, 800 pg, 900 pg, 1 ng, 10 ng, 100 ng, or is in a range defined by any two of these values, for example, 10 pg to 100 pg, 10 pg to 1 ng, 100 pg to 1 ng, 1 ng to 10 ng, 10 ng to 100 ng, etc. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is about 100 pg to about 1 ng. In some embodiments, the nucleic acid sample may comprise an amount of nucleic acid (e.g., genomic DNA) that is more than about 62,5 pg. In some embodiments, additional fragmentation steps, such as sonication or endonuclease digestion, are not included.

[0066] In some embodiments, methods disclosed herein comprise amplification and library preparation in anticipation of downstream parallel sequencing. An assay may include two PGR mastermixes, two thermostable polymerases, two primer mixes and library adaptors. In some embodiments, a sample of DNA may be amplified for a number of cycles by using a first set of amplification primers that comprise target specific regions and non- target specific tag regions and a first PGR mastermix. The tag region can be any sequence, such as a universal tag region, a capture tag region, an amplification tag region, a sequencing tag region, a UMi tag region, and the like. For example, a tag region can be the template for amplification primers utilized in a second or subsequent round of amplification, for example for library preparation. In some embodiments, the methods comprise adding single stranded- binding protein (SSB) to the first amplification products. An aliquot of the first amplified sample can be removed and amplified a second time using a second set of amplification primers that are specific to the tag region, e.g., a universal tag region or an amplification tag region, of the first amplification primers which may comprise of one or more additional tag sequences, such as sequence tags specific for one or more downstream sequencing workflows, and the same or a second PGR mastermix. As such, a library of the original DNA sample is ready for sequencing.

[0067] An alternative method could comprise the first amplification being performed in a small volume (e.g., 15ul) and instead of transferring an aliquot to a new location for a second round of amplification, additional reagents to perform a second round of amplification could be added to the tube.

[0068] Once the library is created, it can be purified and quantitated. In some examples, purification can be performed by processing the sample through a substrate such as AMPURE XP Beads (Beckman Coulter) which serves to purify the DNA fragments away from reaction components. Another method could be the incorporation of a purification moiety, such as a hapten moiety, into the second set of amplification primers. For example, if a biotin was incorporated into one the primers of the second amplification primer set then the library fragments could be capturing using a streptavidin moiety on a bead for example. Utilizing the capture strategy the libraries could also be normalized and quantitated using Bead Based Normalization (BBN). However, libraries can be purified and quantitated, or pooled and quantitated if multiple reactions are being performed, without the use of BBN. For example, libraries could also be quantitated by gel electrophoretic methods, BioAnaiyzer, qPCR, spectrophotometric methods, quantitation kits (e.g., PicoGreen, etc.) and the like as known in the art. Following quantitation, the library can then be sequenced by parallel sequencing.

[0069] in some embodiments, the first set of amplification primers used to amplify a target DNA is provided in such a limited concentration that when an aliquot, of the first amplification reaction is added to a new tube and the reagents from the second amplification reaction are added there is minimal to undetectable carryover amplification resulting from the first set of amplification primers and a cleanup step between the first amplification reaction and the second amplification reaction is not required. In some examples, the concentration of the amplification primers for a first PGR is, is about, or is less than, 0.5nM, Ο.όηΜ, 0.7nM, O.811M, 0.9nM, l .OnM, 1.5nM, 2.0nM, 3,0nM, 4.0nM, 5.0iiM, ό,ΟηΜ, 7.0nM, 8.0nM, 9.0nm ΙΟ.ΟηΜ, I LOnM 12.0nM, or a range between any of these values, for example, 0.5 11M to 1.0 nM, 1.0 nM to 12 nM, 0.8 11M to 1.5 nM, etc. In some embodiments, the concentration of amplification primers for a first PCR is about 0.9 nM to about 10 nM.

[0070] FIG . 2 shows an exemplary workflow of the presently disclosed methods in one embodiment. A target genomic DNA sequence is amplified using a first set of primers comprising a region that flanks the target sequence and amplification tag regions (which may be the same or different) resulting in amplicons comprising the target sequence and tags on both ends. An aliquot of the amplicons from, the first PCR is further amplified using a second set of primers specific to the first tag sequences further comprising sequencing primer sequences (i5 and i7 adapter sequences), thereby generating a library comprising the target DNA sequence flanked by sequences used in parallel sequencing, in this case i5 and i7 sequences are utilized in sequence by synthesis methods popularized by illuniina, Inc.

[0071] An example of an alternative workflow for determining a DNA profile from a sample is described in FIG. 3. In this example, a DNA target is amplified with a first primer pair that comprises sequences that flank the target sequence, non-target tag sequences (the same or different) and further unique molecular identifier sequences or UMIs, which comprise randomized bases. The UMIs can be used, for example, to bioinformatically decrease or eliminate errors that occur during the library preparation processes (e.g., PGR artifacts or misincorporations, etc.). Use of UMIs can be important for DNA profiling, but, are of particular importance for use in helping to eliminate errors when samples are sequenced for criminal casework. In this example, the first, round of amplification is performed for 2 cycles, which is followed by addition of a single stranded binding protein (SSB) and incubation at 37 degree C for 15 min following by a 95 degree C/5 min inactivation which effectively quenches further amplification of the first set of amplification primers during the second round of amplification. Although the mechanism is unknown, it is contemplated that adding the SSB irreversibly binds the single stranded first amplification primers and prevents them from participating in subsequent amplification reactions. Following SSB incubation, a second set of primers comprising sequence tags and a second PGR mix is added resulting in the sequencing library.

Nucleic Acid Library

[0072] Embodiments disclosed herein provide nucleic acid libraries, which may be used for sequencing. In some embodiments, the nucleic acid libraries disclosed herein may comprise a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise at least one tandem repeat sequence flanked by a first pair of tag sequences and at least one single nucleotide polymorphism. (SNP) sequence flanked by a second pair of tag sequences.

[0073] As outlined herein, the size of the nucleic acid molecules may vary greatly- using the methods and compositions disclosed herein. It would be appreciated by those skilled in the art that the nucleic acid molecules amplified from a target sequence comprising a tandem repeat (e.g., STR) may have a large size, while the nucleic acid molecules amplified from a target sequence comprising a SNP may have a small size. For example, the nucleic acid molecules may comprise from less than a hundred nucleotides to hundreds or even thousands of nucleotides. Therefore, the size of the nucleic acid molecules may have a range that is between any two values of about 50 bp, about 60 bp, about 70 bp, about 80 bp, about 90 bp, about 100 bp, about 110 bp, about 120 bp, about 130 bp, about 140 bp, about 150 bp, about 200 bp, about 300 bp, about 400 bp, about 500 bp, about 600 bp, about 700 bp, about 800 bp, about 900 bp, about 1 kb, or more. In some embodiments, the minimal size of the nucleic acid molecules may be a length that is, is about, or is less than, 50 bp, 60 bp, 70 bp, 80 bp, 90 bp, or 100 bp. In some embodiments, the maximum size of the nucleic acid molecules may be a length that is, is about, or is more than, 100 bp, 150 bp, 200 bp, 250 bp, 300 bp, 350 bp, 400 bp, 450 bp, 500 bp, or 1 kb.

[0074] For cluster generation, the library fragments are immobilized on a substrate, for example a slide, which comprises homologous oligonucleotide sequences for capturing and immobilizing the DNA library fragments. The immobilized DNA library fragments are amplified using cluster amplification methodologies as exemplified by the disclosures of US Patent Nos. 7,985,565 and 7,1 15,400, the contents of each of which is incorporated herein by reference in its entirety. The incorporated materials of US Patent Nos. 7,985,565 and 7,1 15,400 describe methods of solid-phase nucleic acid amplification which allow amplification products to be immobilized on a solid support in order to form arrays comprised of clusters or "colonies" of immobilized nucleic acid molecules. Each cluster or colony on such an array is formed from a plurality of identical immobilized polynucleotide strands and a plurality of identical immobilized complementary polynucleotide strands. The arrays so-formed are generally referred to as "clustered arrays". The products of solid-phase amplification reactions such as those described in US Patent Nos. 7,985,565 and 7,115,400 are so-called "bridged" structures formed by annealing of pairs of immobilized polynucleotide strands and immobilized complementary strands, both strands being immobilized on the solid support at the 5' end, preferably via a covalent attachment. Cluster amplification methodologies are examples of methods wherein an immobilized nucleic acid template is used to produce immobilized amplicons. Other suitable methodologies can also

.77 be used to produce immobilized amplicons from immobilized DNA fragments produced according to the methods provided herein. For example one or more clusters or colonies can be formed via solid-phase PCR whether one or both primers of each pair of amplification primers are immobilized. However, the methods described herein are not limited to any particular sequencing preparation methodology or sequencing platform and can be amenable to other parallel sequencing platform preparation methods and associated sequencing platforms.

Primers

[0075] Embodiments disclosed herein provide a plurality of primers that specifically hybridize to at least one short target sequence and at, least, one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single multiplex reaction results in at least one short amplification product and at least one long amplification product, wherein each of the plurality of primers comprises one or more tag sequences. Further disclosed herein is a plurality of primers that have the sequences set forth in Tables 1 -2.

[0076] For multiplex amplification of large target sequence (e.g., STRs, ITRs) and small target, sequence (e.g., SNPs), primers are designed that would allow for balanced amplification across ail the target types. The methods and compositions disclosed herein may be used to amplify multiple tandem repeat target sequences in a single multiplex reaction. For example, the plurality of primers may specifically hybridize to a number of tandem repeat sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 60 tandem repeat, sequences. The methods and compositions disclosed herein may be used to amplify multiple SNP target sequences in a single reaction. For example, the plurality of primers may specifically hybridize to a number of SNP sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 30 SNP sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 50 SNP sequences.

[0077] it was discovered during experimentation that the short SNP target sequences preferentially amplified over the longer STR target sequences when using primers that were designing following established criteria and wisdom for successful primer design. Further, at least in the sequence by synthesis workflow where clusters are generated and the clusters are themselves sequenced (for example, when following sequence by synthesis (SBS, disclosed herein elsewhere) associated with the Alumina, Inc. sequencers) preferential cluster amplification of the shorter library SNP fragments also occurred. To overcome these two biases, a new strategy was needed for primer design that would allow for balanced amplification between the short SNP target sequences and the long STR target sequences.

[0078] One of the strategies included designing primers for STR amplification. With STRs, the repeated sequences are often embedded within larger repeated regions; therefore designing specific primers for STR amplification can be problematic. Further, STRs and their flanking regions are oftentimes AT rich. In one instance, primers were designed to the problematic regions using a design strategy contrary to conventional and well established PGR design criteria. The established criteria for PGR primer design states that, among other criteria, 1) optimal length for primers is 18-22 nucleotides, 2) the Tm should be in the range of 55-58 degrees C, 3) GC content should be around 40-60%, 4) and repeated AT dinucleotide regions should be avoided, with <4 dinucleotide AT repeats being the maximum. Primers were designed that were longer than typical PGR. primers, for example 23-35 nucleotides long instead of 18-22 nucleotides, they had low melting temperatures (Tm), for example around 54 degrees C instead of around 58 degrees C, and the primers were AT rich, three parameters that conventional established PGR criteria teach should be avoided for optimal primer design. In effect, non-optimal primers were designed. Surprisingly, it was discovered that these long, AT rich, low Tm primers actually multiplexed the STRs better than the short, high Tm low AT containing primers. Without being bound to any theory, it is contemplated that the shorter primers that were designed following established PGR design criteria might form dimers that had high melting temperatures and thus formed dimers efficiently under normal PCR conditions, whereas the longer, low Tm primers might form dimers u der really low Tm and thus would not be stable for dimer formation, thereby allowing for increased participation of the longer, low Tm primers under normal amplification conditions compared to the short, high Tm primers (e.g., 18-22 nucleotides, Tm of 60 degrees C, 50% GC content).

[0079] The longer, low Tm, AT rich primers for STR amplification were then multiplexed with the conventionally designed, high Tm shorter primers that targeted SNPs. However, the multiplex amplification reactions were once again unsuccessful in providing a balanced amplification of both STRs and SNPs in one multiplex reaction. It was contemplated that perhaps applying the unconventional primer design to amplify non- problematic targets, for example to amplify the SNP targets, might yield successful multiplex amplifications. As such, the same criteria used to design non-optimal primers for STRs were applied to primer design for SNPs (long, low Tm, AT rich). Surprisingly, the new designed primers resulted in better balance between amplification of STRs and SNPs in a multiplex reaction.

[0080] FIG .4 shows examples of the interplay between the conventional and unconventional designed primers in a multiplex reaction. In FIG. 4A a multiplex reaction of 10 SNP targets shows expected amplification in the desired range of around 200-350 bp for the library. The primers used to amplify the 10 SNPs in the multiplex were designed to be longer, have lower Tm and be more AT rich that is advised by established PCR primer design criteria. When an 1 1 ^* primer pair is designed using the established PCR design criteria, that is the primers are short, have high Tm and are not AT rich, and added to the 10 pairs the resulting multiplex shows non-specific amplification of the target DNA. As seen in FIG. 4B and 4D, the addition of an ^dl conventionally designed primer pair interferes with the 10 plex of unconventional primer pairs and results in an unsuccessful multiplex amplification of targeted SNPs. However, the addition of an l l^ffi primer pair that is also unconventionally designed following the same criteria as the 10 plex of primer pairs results in the successful amplification of the SNP targets (FIG. 4C).

[0081 ] Accordingly, in some embodiments, each of the plurality of primers has a low melting temperature, e.g., less than 60 degrees C or about 50 degrees C to about 60 degrees C, and/or has a length of at least 24 nucleotides, e.g., about 24 nucleotides to about 38 nucleotides. In some embodiments, each of the plurality of primers comprises a bomopolymer nucleotide sequence.

[0082] in some examples, the unconventionally designed primers comprise sequences that flank the targeted STRs and S Ps and additional non-template sequences. The additional sequences can be, for example tag sequences that serve a purpose during library preparation or sequencing methodologies. For example, a tag sequence can be a capture sequence such as a hapten moiety than can be captured by an immobilized partner moiety for purifying library fragments. An example of a hapten moiety is biotin which can be captured by streptavidin for isolated librar}' fragments from reaction components and the like. A tag sequence could also be an amplification sequence, for example that is complementary to an amplification primer and is used in one or more amplification reactions. FIG. 2 and 3 show examples of tag sequences that are used in a second round of amplification following a first round of amplification. A tag sequence could also be a sequence tag. FIG. 2 and 3 also show examples of sequence tags, i5 adapter and i7 adapter are used in sequencing as hybridization, cluster generation and sequencing primers during the sequence by synthesis reactions as described herein. Another example of a tag sequence is a unique molecular identifier, or UMI, as shown in FIG. 3.

[0083] A UMI comprises a random stretch of nucleotides that can be used during sequencing to correct for PGR and sequencing errors, thereby adding an additional layer of error correction to sequencing results. UMIs could be from, for example 3-10 nucleotides long, however the number will depend on the amount of input DNA. For example, if Ing DNA is used to target around 250 sites, then it is anticipated that approximately 350 copies x 250 targets would be needed, so approximately 90,000 different UMIs. If more DNA is utilized, for example lOng, then approximately 1 million different UMIs could be needed. All PGR. duplicates from the same PGR reaction would have the same UMI sequence, as such the duplicates can be compared and any errors in the sequence such as single base substitutions, deletions, insertions (i.e., stutter in PGR) can be excluded from the sequencing results bioinformatically. Unique molecular identifiers can also be used in analysis for a mixed sample. Mixed samples, for example a female DNA sample that is contaminated with male DNA, can be deconvoiuted to report both the female and male DNA contributions using UMI sequences. For example, there could be a total of four different repeated numbers for two mixed DNAs; however there could be less than four if the mixture of two samples shares alleles at a particular locus. These shared alleles can be distinguished and approximate percentages determined using the UMis for determining the number of different alleles in the initial population of DNA molecules. For example, the initial molecules could be counted and if a minor contributor was present at, for example 5%, then 5% of the UMis would identify one genotype and 95% would identify a second genotype. After PGR, if one of the alleles (or perhaps more) was biased upon amplification then that 5:95 ratio would not be seen. However, using UMis a biased ratio could be corrected after PGR duplicates are condensed using UMI detection and correction. This is important when trying to differentiate from a stutter artifact from PCR and a true minor contributor.

[0084] A primer of the present methods can comprise one or more tag sequences. The tag sequences can be one or more of primer sequences that are not homologous to the target sequence, but, for example can be used as templates for one or more amplification reactions. The tag sequence can be a capture sequence, for example a hapten sequence such as biotin that can be used to purify amplicons away from reaction components. The tag sequences can be sequences such as adaptor sequences that are advantageous for capturing the library amplicons on a substrate for example for bridge amplification in anticipation of sequence by synthesis technologies as described herein. Further, tag sequences can be unique molecular identifier tags of typically between, for example, 3-10 nucleotides comprised of a randomized stretch of nucleotides that can be used for error correction during library preparation and/or sequencing methods.

[0085] Additionally, it is advantageous for a multiplexed PCR reaction to contain oligonucleotide primers to substantially all of the targets pooled together into one mix. However, as disclosed herein, the oligonucleotides are uncharacteristically longer than primers designed using traditional parameters. Further addition of tag sequences to the primers, such as the addition of UMis that append a gene target, specific sequence create still longer primer sequences. In some embodiments, glycine betaine (approximately 1 .5M) may be added to the plurality of primers. For example, in some embodiments the amplification

-77- buffers used in amplification reactions with unconventional primers as disclosed herein comprise a betaine concentration that is, is about, or is more than, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1 M, 1.2 M, 1.3 M, 5.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, or a range between any two of these values, for example, from 500 mM to 2 M, from 1 M to 1 .5 M, etc. As such, a primer mix as described herein supplemented with betaine, for example at approximately 1 .5M, would be advantageous when practicing methods of the present disclosure. In some embodiments, glycerol may be added to the plurality of primers. For example, in some embodiments the amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a glycerol concentration that is, is about, or is more than, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, 1 M, 1.2 M, 1.3 M, 1.4 M, 1.5 M, 1.6 M, 1.7 M, 1.8 M, 1.9 M, 2 M, 3 M, 4 M, 5 M, 6 M, 7 M, 8 M, 9 M, 10 M, or a range between any two of these values, for example, from 500 mM to 2 M, from 1 M to 1.5 M, etc. As such, a primer mix as described herein supplemented with glycerol, for example at approximately 1.5M, would be advantageous when practicing methods of the present disclosure.

|0086] In some embodiments, buffers associated with unconventional primer design used in amplification methods of the present disclosure may also be modified. For example, in some embodiments the salt concentrations, such as KC1, LiCl, NaCi, or a combination thereof, of the amplification buffer are increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a C1 concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KG concentration that is about 145 mM, In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a LiCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a LiCl concentration that is about 145 mM. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a NaCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 110 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM. 200 mM, 250mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a NaCl concentration that is about 145 mM.

[0087] In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein may comprise MgS0₄, MgCfe, or a combination thereof.

Kits

[0088] Embodiments disclosed herein provide kits comprising at least one container means, wherein the at least one container means comprises a plurality of primers as disclosed herein. In some embodiments, the container means may be a tube, a well, a microliter plate, etc. In some embodiments, the plurality of primers may specifically hybridize to a number of tandem repeat sequences that is, is about, or is more than 4, 6, 8, 10, 12, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 24 tandem repeat sequences. In some embodiments, the plurality of primers may specifically hybridize to at, least 60 tandem repeat sequences. The methods and compositions disclosed herein may be used to amplify multiple SNP target sequences in a single reaction. For example, the plurality of primers may specifically hybridize to a number of SNP sequences that is, is about, or is more than 4, 6, 8, 10, 52, 14, 16, 18, 24, 30, 40, 50, 60, 70, 80, 90, 100, or a range between any of the two values, such as 4 to 12, 10 to 24, 30 to 100, etc. In some embodiments, the plurality of primers may specifically hybridize to at least 30 SNP sequences. In some embodiments, the plurality of primers may specifically hybridize to at least 50 SNP sequences.

[0089] In some embodiments, the at least one container means comprises an amplification buffer. In some embodiments, buffers associated with unconventional primer design used in amplification methods of the present disclosure may also be modified. For example, in some embodiments the salt concentrations, such as KG, LiCl, NaCl, or a combination thereof, of the amplification buffer are increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KG, NaCl or LiCl concentration that is, is about, or is more than, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 1 10 mM, 120 mM, 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, 200 mM, 250mM, 300 mM, 400 mM, 500 mM, or a range between any two of these values, for example, from 60 mM to 200 mM, from 100 mM to 250 mM, etc. In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein comprise a KG, NaCl or LiCl concentration that is about 145 mM.

[0090] In some embodiments, amplification buffers used in amplification reactions with unconventional primers as disclosed herein may comprise MgSO.*, MgCfe, or a combination thereof.

Sequencing Methods

[0091 ] The present methods are not limited to any particular sequencing platform, however are being exemplified here in regards to SBS, or sequence by synthesis, type of parallel sequencing. Particularly applicable techniques are those wherein nucleic acids are attached at fixed locations in an array such that their relative positions do not change and wherein the array is repeatedly imaged. Examples in which images are obtained in different color channels, for example, coinciding with different labels used to distinguish one nucleotide base type from another are particularly applicable.

[0092] SBS techniques generally involve the enzymatic extension of a nascent nucleic acid strand through the iterative addition of nucleotides against a template strand. In traditional methods of SBS, a single nucleotide monomer may be provided to a target nucleotide in the presence of a polymerase in each delivery. However, in the methods described herein, more than one type of nucleotide monomer can be provided to a target nucleic acid in the presence of a polymerase in a delivery.

[0093] SBS techniques can utilize nucleotide monomers that have a. label moiety or those that lack a label moiety. Accordingly, incorporation events can be detected based on a characteristic of the label, such as fluorescence of the label; a. characteristic of the nucleotide monomer such as molecular weight or charge; a byproduct of incorporation of the nucleotide, such as release of pyrophosphate: or the like. In some examples where two or more different nucleotides are present in a sequencing reagent, the different nucleotides can be distinguishable from each other, or alternatively, the two or more different labels can be the indistinguishable under the detection techniques being used. For example, the different nucleotides present in a sequencing reagent can have different labels and they can be distinguished using appropriate optics as exemplified by the sequencing methods developed by Soiexa (now Illumina, Inc.).

[0094] Some examples include pyrosequencing techniques, Pyrosequencing detects the release of inorganic pyrophosphate (PPi) as particular nucleotides are incorporated into the nascent strand (Ronaglii, M., Karamoliamed, S., Pettersson, B., Uhlen, M. and Nyren, P. (1996) "Real-time DNA sequencing using detection of pyrophosphate release." Analytical Biochemistry 242(1), 84-9; Ronaglii, M. (2001 ) "Pyrosequencing sheds light on DNA sequencing." Genome Res. 1 1 (1 ), 3-1 1 ; Ronaghi, M, Uhlen, M. and Nyren, P. ( 1998) "A sequencing method based on real-time pyrophosphate." Science 281(5375), 363; U.S. Pat. No. 6,210,891 ; U.S. Pat. No. 6,258,568 and U.S. Pat. No. 6,274,320, the disclosures of which are incorporated herein by reference in their entireties). In pyrosequencing, released PPi can be detected by being immediately converted to adenosine triphosphate (ATP) by ATP sulfurylase, and the level of ATP generated is detected via luciferase-produced photons. The nucleic acids to be sequenced can be attached to features in an array and the array can be imaged to capture the chemiluminscent signals that are produced due to incorporation of a nucleotides at the features of the array. An image can be obtained after the array is treated with a particular nucleotide type (e.g. A, T, C or G). Images obtained after addition of each nucleotide type will differ with regard to which features in the array are detected. These differences in the image reflect the different sequence content of the features on the array. However, the relative locations of each feature will remain unchanged in the images. The images can be stored, processed and analyzed using the methods set forth herein. For example, images obtained after treatment of the array with each different nucleotide type can be handled in the same way as exemplified herein for images obtained from different detection channels for reversible terminator-based sequencing methods.

[0095] in another example of SBS, cycle sequencing is accomplished by stepwise addition of reversible terminator nucleotides containing, for example, a cleavable or photobieachable dye label as described, for example, in WO 04/018497 and U.S. Pat. No. 7,057,026, the disclosures of which are incorporated herein by reference. This approach is being commercialized by Solexa (now Illumina Inc.), and is also described in WO 91 /06678 and W 07/123,744, each of which is incorporated herein by reference. The availability of fluorescently-labeled terminators in which both the termination can be reversed and the fluorescent label cleaved facilitates efficient cyclic reversible termination (CRT) sequencing. Polymerases can also be co-engineered to efficiently incorporate and extend from these modified nucleotides. Additional exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Patent Application Publication No. 2007/0166705, U.S. Patent Application Publication No. 2006/0188901 , U.S. Pat. No. 7,057,026, U.S. Patent Application Publication No. 2006/0240439, U.S. Patent Application Publication No. 2006/0281109, PCT Publication No. WO 05/065814, U.S. Patent Application Publication No. 2005/0100900, PCT Publication No. WO 06/064199, PCT Publication No. WO 07/010,251, U.S. Patent Application Publication No. 2012/0270305 and U.S. Patent Application Publication No. 2013/0260372, the disclosures of which are incorporated herein by reference in their entireties. [0096] Some examples can utilize detection of four different nucleotides using fewer than four different labels. For example, SBS can be performed utilizing methods and systems described in the incorporated materials of U.S. Patent Application Publication No. 2013/0079232. As a first example, a pair of nucleotide types can be detected at the same wavelength, but distinguished based on a difference in intensity for one member of the pair compared to the other, or based on a change to one member of the pair (e.g. via chemical modification, photochemical modification or physical modification) that causes apparent signal to appear or disappear compared to the signal detected for the other member of the pair. As a second example, three of four different nucleotide types can be detected under particular conditions while a fourth nucleotide type lacks a label that is detectable under those conditions, or is minimally detected under those conditions (e.g., minimal detection due to background fluorescence, etc). Incorporation of the first three nucleotide types into a nucleic acid can be determined based on presence of their respective signals and incorporation of the fourth nucleotide type into the nucleic acid can be determined based on absence or minimal detection of any signal. As a third example, one nucleotide type can include label(s) that are detected in two different channels, whereas other nucleotide types are detected in no more than one of the channels. The aforementioned three exemplary configurations are not considered mutually exclusive and can be used in various combinations. An exemplary embodiment that combines all three examples, is a fluorescent-based SBS method that uses a first nucleotide type that is detected in a first channel (e.g. dATP having a label that is detected in the first channel when excited by a first excitation wavelength), a. second nucleotide type that is detected in a second channel (e.g. dCTP having a label that is detected in the second channel when excited by a second excitation wavelength), a third nucleotide type that is detected in both the first and the second channel (e.g. dTTP having at least one label that is detected in both channels when excited by the first and/or second excitation wavelength) and a fourth nucleotide type that lacks a label that is not, or minimally, detected in either channel (e.g. dGTP having no label).

[0097] Further, as described in the incorporated materials of U.S. Patent Application Publication No. 2013/0079232, sequencing data can be obtained using a single channel. In such so-called one-dye sequencing approaches, the first nucleotide type is labeled but the label is removed after the first image is generated, and the second nucleotide type is labeled only after a first image is generated. The third nucleotide type retains its label in both the first and second images, and the fourth nucleotide type remains unlabeled in both images.

[0098] Some examples can utilize sequencing by ligation techniques. Such techniques utilize DNA ligase to incorporate oligonucleotides and identify the incorporation of such oligonucleotides. The oligonucleotides typically have different labels that are correlated with the identity of a particular nucleotide in a sequence to which the oligonucleotides hybridize. As with other SBS methods, images can be obtained following treatment, of an array of nucleic acid features with the labeled sequencing reagents. Each image will show nucleic acid features that have incorporated labels of a particular type. Different features will be present or absent in the different images due the different sequence content of each feature, but the relative position of the features will remain unchanged in the images. Images obtained from ligation-based sequencing methods can be stored, processed and analyzed as set forth herein. Exemplary SBS systems and methods which can be utilized with the methods and systems described herein are described in U.S. Pat. No. 6,969,488, U.S. Pat. No. 6,172,21 8, and U.S. Pat. No. 6,306,597, the disclosures of which are incorporated herein by reference in their entireties.

[0099] Some examples can utilize nanopore sequencing (Deamer, D. W. & Akeson, M. "Nanopores and nucleic acids: prospects for ultrarapid sequencing." Trends Biotechnol. 18, 147- 151 (2000); Deamer, D. and D. Branton, "Characterization of nucleic acids by nanopore analysis". Acc. Chem. Res. 35:817-825 (2002); Li, J., M. Gershow, D. Stein, E. Brandin, and J. A. Golovchenko, "DNA molecules and configurations in a solid- state nanopore microscope" Nat. Mater. 2:61 1 -615 (2003), the disclosures of which are incorporated herein by reference in their entireties). In such embodiments, the target nucleic acid passes through a nanopore. The nanopore can be a synthetic pore or biological membrane protein, such as a-hemolysin. As the target nucleic acid passes through the nanopore, each base-pair can be identified by measuring fluctuations in the electrical conductance of the pore. (U.S. Pat. No. 7,001 ,792; Soni, G. V. & Meller, "A. Progress toward ultrafast DNA sequencing using solid-state nanopores." Clin. Chem. 53, 1996-2001 (2007); Healy, K. "Nanopore -based single-molecule DNA analysis." Nanomed. 2, 459-481 (2007); Cockroft, S, L,, Chu, J., Amorin, M. & Ghadiri, M. R. "A single-molecule nanopore device detects DNA polymerase activity with single-nueleotide resolution." J. Am. Chem. Soc. 130, 818-820 (2008), the disclosures of which are incorporated herein by reference in their entireties). Data obtained from nanopore sequencing can be stored, processed and analyzed as set forth herein. In particular, the data can be treated as an image in accordance with the exemplary treatment of optical images and other images that is set forth herei .

[0100] Some examples can utilize methods involving the real-time monitoring of DNA polymerase activity. Nucleotide incorporations can be detected through fluorescence resonance energy transfer (FRET) interactions between a fiuorophore -bearing polymerase and γ-phosphate-labeled nucleotides as described, for example, in U.S. Pat. No. 7,329,492 and U.S. Pat. No. 7,211,414 (each of which is incorporated herein by reference) or nucleotide incorporations can be detected with zero-mode waveguides as described, for example, in U.S. Pat. No. 7,315,019 (which is incorporated herein by reference) and using fluorescent nucleotide analogs and engineered polymerases as described, for example, in U.S. Pat. No. 7,405,281 and U.S. Patent Application Publication No. 2008/0108082 (each of which is incorporated herein by reference). The illumination can be restricted to a zeptoliter-scale volume around a surface-tethered polymerase such that incorporation of fluorescently labeled nucleotides can be observed with low background (Levene, M. j. et al. "Zero-mode waveguides for single-molecule analysis at high concentrations." Science 299, 682-686 (2003); Lundquist, P. M. et al. "Parallel confocal detection of single molecules in real time." Opt. Lett. 33, 1026-1028 (2008); Korlach, J. et al. "Selective aluminum passivation for targeted immobilization of single DNA polymerase molecules in zero-mode waveguide nano structures." Proc. Natl. Acad. Sci. USA 105, 1176-1181 (2008), the disclosures of which are incorporated herein by reference in their entireties). Images obtained from such methods can be stored, processed and analyzed as set forth herein.

[0101] Some SBS embodiments include detection of a proton released upon incorporation of a nucleotide into an extension product. For example, sequencing based on detection of released protons can use an electrical detector and associated techniques that are commercially available from Ion Torrent (Guilford, CT, a Life Technologies subsidiary) or sequencing methods and systems described in US 2009/0026082 Al; US 2009/0127589 Al ; US 201 0/0137143 Al ; or US 2010/0282617 Al , each of which is incorporated herein by reference. Methods set forth herei for amplifying target nucleic acids using kinetic exclusion can be readily applied to substrates used for detecting protons. More specifically, methods set forth herein can be used to produce clonal populations of amplicons that are used to detect protons.

[0102] The above SBS methods can be advantageously carried out in multiplex formats such that multiple different target nucleic acids are manipulated simultaneously. In particular embodiments, different target nucleic acids can be treated in a common reaction vessel or on a surface of a particular substrate. This allows convenient delivery of sequencing reagents, removal of unreacted reagents and detection of incorporation events in a multiplex manner. In embodiments using surface-bound target nucleic acids, the target nucleic acids can be in an array format. In an array format, the target nucleic acids can be typically bound to a surface in a spatially distinguishable manner. The target nucleic acids can be bound by direct covalent attachment, attachment to a bead or other particle or binding to a polymerase or other molecule that is attached to the surface. The array can include a single copy of a target nucleic acid at each site (also referred to as a feature) or multiple copies having the same sequence can be present at each site or feature. Multiple copies can be produced by amplification methods such as, bridge amplification or emulsion PGR as described in further detail below.

[0103] The methods of the present disclosure utilize the Ill mina, Inc. technology for sequencing the DNA profile libraries created by practicing the methods described herein. The MiSeq sequencing instrument was used for clustering and sequencing for the examples described herein. However, as previously stated and as understood by a skilled artisan, the present methods are not limited by the type of sequencing platform used.

EXAMPLES

[0104] The fol lowing examples disclose several methods and materials for DNA profiling. These methods and materials can be modified while maintaining the spirit and scope of the invention. Such modifications will become apparent to those skilled in the art from a consideration of this disclosure or practice of the methods disclosed here. Consequently, it is not intended that these methods or materials be limited to the specific examples disclosed herein, but that it cover all modifications and alternatives that fall within the scope and spirit of the present disclosure.

Example ΐ -Unconventional primer design

[0105] The computer design program DesignStudio from illumina, Inc. (San Diego, CA) was modified and used for primer design. A. skilled artisan would of course understand that alternative primer design programs such as Primer3 can also be used and the default, parameters reset to mimic the intent of the modified parameters for primer design. The settings are typically reset in the config.xml file that comes with the software, however this may differ when using different, software and consulting the specific materials for accessing default parameters for each software is typical practice. The following parameters can be reset in the primer design software:

1) The desired minimum length amplicon reset to >60<

2) The desired maximum length amplicon reset to >120<

3) The tight candidate spacing reset to >3< (default is 3()bp)

4) The %GC Max probe reset to >60< to allow for increased number of AT rich repeat stretches

5) The mean Tm reset to >57< (default 59C) to lower the mean Tm

6) The maximum Tm reset to >60< (default 71 )

7) The minimum Tm reset to >51< (default 55)

8) The mean probe length reset to >28< (default 27)

9) The maximum probe length reset to >38< (default 30)

10) The minimum probe length reset to >25< (default 22)

[0106] For designing the SNP primers, the range to target for the 3' end of the primer was set to "small" to keep the primers around l p away for the targeted S^'NP. Once all the parameters are reset, the primer design program can be run on the sequence to determine the primer pair candidates that fall under the new parameters. For example, a user of the software can generate a targets list that tells the software where to look in the genome for designing the primers. In the present example, the targeted regions were copy and pasted into the graphic user interface application which the DesignStudio software used to orient and target primer design. Once the targeted regions were input into the program, the program directed to Create a Design File to start the tool and create the primer designs. In the present example, the main output was a ,txt file that included the primer sequences and/or some of the regions contained failures and were "undesignable", at which point the targeted sequences needed to he redefined and rerun. The software used in this experiment provided the designed primers that were mapped onto the sequence that was specified as the targeted region. Following the reset parameters, primers were designed that did not follow the conventional criteria for primer design for amplification; however which allowed for the multiplex amplification of long STRs and short SNPs.

[0107] Examples of designed ST targeted primers advantageous in methods disclosed herein include those listed in Table 1. Examples of SNP targeted primers advantageous in methods disclosed herein include those listed in Table 2.

Table 1-STR targeted primer without, tags and amplicon sizes

D61043 l Sm TCTGTGGTTCTCCAGCTTAC

TPOX Fl T CTTAGGGAACCCTCACTGAATG 77

ΪΡΟΧ R l Sm GTCCITGTCAGCGTTTATiTGC

D13S317 F^'2 T TTGGGT GAGCCATAGGCAG 162

D13S317 R2 Sm GCATCCGTGACTCTCTGGAC

D21S11 Fl T GTTATGGGACTTTTCTCAGTCTCCAT 226

D21S11 R3 Sm GAGACTAATAGGAGGTAGATAGACTGG

D12S391 Fl T GAGACTGTATTAGTAAGGCTTCTC 253

D12S391 R2 Sm CCTGGACTGAGCCATGCTCC

D1S1656 F2 T CAGTCCTGTGTTAGTCAGGATTC 173

D1S1656 Rl Sm TCAAGGGTCAACTGTGTGATGT

D9S1122 F3 T i l U AAAGC ! ! C I AG 1 1 1 AC I 120

D9S1122 R2 Sm TTGCTTATTTGTGGGGGTATTTCA

PentaE Fl 1^" AAGAATTCTCTTATTTGGG TATTAATTG 362

PentaE^" Rl Sm AAA^'ITGTGGACAGGTGCGGT

D17S1301_F2_T CCATGTAAAAATACATGCATGTG nTATTTATAC 142

D17S1301_R2_Sm TGATTAAAAAGAATGAAGGTAAAAATGTGTATAAC

D2S441 F2 T CCAAATGTTTATGATTAATCTTTTAAATTGGAGC 160

D2S441 R3 Sm GTAACAAGGGCTACAGGAATCATGAG

D4S2408. F3 J TCATCCACTGAAATGACTGAAAAATAG 102

D4S2408 R9 Sm AGGTAC ATAACAGTTC AATAG AAAG

D2S1338 F2 T GAGTTATTCAGTAAGTTAAAGGATTGCAG 162

D2S1338 R2 Sm GGGAGCCAGTGGATTTGGAAACAG

PentaD F3 T GCATGGTGAGGCTGAAGTAG 268

PentaD Rl Sm CTAACCTATGGTCAT AACG ATTTTT

vWA F3 T GATGATAAGAATAATCAGTATGTGACITGG 160 vWA R3 Sm ATAGG^'ITAG ATAGAG AT AGGACAGATGATA

SE33 Fl Ϊ CCCTACCGCTATAGTAACTTGC 380

SE33 R2 Sm CACGTCTGTAATTCCAGCTCCTA

D20S482_F3_r GGAAGCGTGTACTAGAGTTCTTCAG 145

D20S482_R2_Sm GGACAGCCTCCATATCCACATG

DXS10074..F1. T TTCCTACTGCCCCACCTTTATTG 212

DXS10074..Rl .sm TTTATGGTCTCAGTGC CCCTC AG A

DXS101.03 F sm TCATAATCACATATCACATGAGC 177

DXS101.03 Rl T AAACAGAACCAGGGGAATGAA

DXS10135_F1_T TGAAACTAAAGTCAAATGGGGCTAC 268

DXS10135_Rl_sm TAAGGGGTGACACCTCT^'CTGGATA

DXS8377_F2_sm CCCAGCCTACATCTACCACrrCATG 276

DXS8377_R2_T Ci^'AATGTTCGTATGGACC TTGGAAAGC

DXS7423_Fl_sm GTCTCCAGTACCCAGCTAGCTTAG 191

DXS7423_R1_1^" TCTCCCAACCTGCCCTTTATCA

DXS8378_Fl_sm TTTGGGCTGACACAGTGGCT 442

DXS8378 Rl T TTGATCAACACAGGAGGTTTGACC

HPRTB..Fl. sm TATACCACTTTGATGTTGACACTAGTTTAC 213

HPRTB R l T CCTGTCTATGGTCTCGATTCAAT

DXS10148_F3_sm TGCATGACAGAGGGAGATTCT 256

DXS10148_R3_T AGAGGGGAAATAGTAGAATGAGGATG

DXS7332_F3_sm GCCAAACTCTA TVAGTC AACGTTC 204

DXS7132_R4_T CTGGTTCT^'CTAGCTCACATACAGT

DYF387Slab F2 T TTTACCCCTAACAAGAAAAAAAGAAGAA 227,231

DYF387Slab R2 Sm CAGTGTGAG AAGTGTG AGAAGTGC

DYS385a b Fl T GACACCATGCCAAACAACAAC 260,248

DYS385a b Rl Sm ATCTATCTATTCCAATTACATAGTCC

DYS389I ii F3 T TCATTATAC CTACTTCTGTATC C A ACTCTC 183,303

DYS389I ii R3 Sm GGAACACAATTATCCCTGAGTAGCAG 81 DYS390 F2 T GGTAGCATAATAGAAATTTTATGAGTGGG 3 8

82 DYS390 R2 Sm GAAGACAGACTTCAATATCACAGAACATCG

83 DYS391 Fl T G^'i GTA^'i CiAVrCA^'iT C AA^'i CAVACACCC 143

84 DYS391 Rl Sm CTCCCTGG TVGCAAGCAATVGCC

85 DYS438 Fl T CCAAAAITAGTGGGGAATAGTTGAAC 149

86 DYS438 R2 Sm GTCGAGATCACACCATTGCATTTC

87 DYS439 Fl T GCCTGGCTTGGAATTCTTTTACCC 195

88 DYS439 Rl Sm TTTAAGTCTTTAATCTATCTTGAATTAATAGATTC

89 DYS481 Fl T CTTTAAGAGGAGTCTGCTAAAAGGAATG 144

90 DYS481 R3 Sm TCACCAGAAGGTTGCAAGAC

1 DYS505 Fl T TCTGGCGAAGTAACCCAAAC 174

92 DYS505 R l Sm TCGAGTCAGTTCACCAGAAGG

93 DYS522 F2 T GGAACCAGTGAGAGCCG 306

94 DYS522 R2 Sm CTCAGAGTGCTGAACCCAG

95 DYS533 F2 1^" GTATTTA VCATGATCAGTTCrTAACT^'CAACC 206

CTACCTAATATTTATCTATATCATTCTAATTATGTCTCTT

96 DYS533 R2 Sm C

97 DYS549 Fl T U U AAAGG ! ! ! ! ! ! ! ! Gb ! GG 1 AAb 222

98 DYS549 R l Sm GATTAATACAACAAAAATTTGGTAATCTGAAA

99 DYS570 Fl T C A ACCTA AG CTG A A ATG C AG ATATTC 170

100 DYS570 Rl Sm GTTATGAAACGTAAAATGAATGATGACF^'AG

101 DYS576 F2 V GCAGTCTCAITTCCTGGAGATGAAGG 191

102 DYS576 Rl Sm CITGGGCTGAGGAGTrCAATC

103 DYS612 F2 Ϊ GCCAGTAAGAATAAAATTACAGCATGAAG 287

104 DYS612 R2 Sm GAATAATCTACCAGCAACAATGGCT

105 DYS635 F4 T TGCCCAATGGAATGCTCTCT 274

106 DYS635 R2 Sm GCTCCATCTCAAACAACAAAAACACAAAAAATG

107 DYS643 F2 T GGGTCATTGAACCTCATGCTCTG 170

108 DYS643_Rl_Sm CCCCCCAAAATTCTACTGAAGTAAA

109 Y GATAH4 F2 T TA ACAGG ATAAATCACCTATCTATGTAT 175

110 Y GATAH4 R2 Sm GCTGAGGAGAATTTCCAAATTTA

Table 2-SNP targeted primers

129 rsl382.387 iSNPI T F G (.1 I AI lUXAIfa! IGIG!A

130 r.\ : ;.>! iSNPI T F CACTCTTCTGAATCCTGGTCAACAAC

131 rsl454361_iSNPIJ^"_F CAAG^"i^"i^"Al^"ATCAl^"AGAGTCl^"ACGACCCC

132 rsl463729 iSNPI T F CTGCAACTATCAGTCrCTGCCClTATTC

133 iSNPI T F GATGTGTCTCAAACVGTTTA VGTGAGG

134 iSNPi T F GAACTCATTTATCCAGAGACCTGTTCTC rsl523537 iSNPi T F CATAATACAACCTGTCITFGGAGITACT rsl528460 iSNPi T F GTGACCAGTAGTTCTATGAGCAAGTATG

137 rs iSNPI T F LACA 11 G 1 A 1 GG i i i i i AGGCACCA 1 G

138 rsl736442 iSNPi T F CTAATAAGTGGGACAGTTAAGAGAAGGC

139 IS1821380 iSNPI T F CAAGACAAGCGATTGAAAGAAGTGGAT iSNPI T F (.!.11 ti ! CAA ICl l ltl A CA AGGti 1 AA

141 rsl979255 iSNPI T F GAATCATAGCTTGTGTTGGTCAGGG

142 iSNPI T F GAAlTACAAGTATTTGCATCCCAGCCr iSNPI T F GACCAAC1TGGCTTTAACAGATGCAAAT

144 iSNPI T F2 TCCTTACCnTAAGACTIT CCTATTTG

145 rs20S6277JSNPI.T..F2 CATTATCTCGTCATACTTCCCTGTCTTG

146 rs2076848 iSNPI T F GCATCAAATTCACCAGTGAAATTATTGA

147 rs2107612 iSNPI T F ATGAGTACATTATTCAACTGTTTTGGAG

148 rs2111980 iSNPi T F CAGCCA IGt tti! AAACA M i l ! ACGG 1

149 rs214955 iSNP! T F GCACATTCTAAGAACTGGTGATTCTATC

150 rs221956 iSNP! T F GCTAGAAAAAGCTGAGATAGCTGTGAAG

151 rs2342.747 iS P! T F C I I AAG I A! ! ! ! !GI I ICCC

152 rs2399332 iS P! T F CTGGACACCAGACCAAAAACAAATAACC rs251934_iSNP!J^"_F GTA ATT AG AG G GC AGTG AGG CTITTA A

154 rs279844 iSNP! T F CTCCAGAAGCTACTGGGATATTAATTAG

155 rs2S30795 iSNPI T F TGAGCCAAATCAGCAATATAATAGGACT rs2831700 iSNPi T F CCTAGAACCACAATTATCTGTCTTTGGC

157 rs2920816 iSNPi T F2 CCATTGATTCTCTACAGTTCTGCAGGTA

158 rs321198 iSNP! T F CTCCACACTTTATACAGGTGAAATCTGA

159 rs33SS32 iSNPI T F A! 111 !CIC!CU IC!GICICAC I IC rs354439 iSNPI T F GCI ICICI 1 IL CI 1 A! G 1 A! 1 L 1 C^■ C

161 rs37S0962 iSNPi T F GGCTTTTGAAGAAAAACACTAACCTGTC

162 -S430046 iSNPI T F i.ACC ! A 1 GGGC 1 L 11 i ! A L 1 CC iSNPI T F CATTTGATAGCCATTTGGGTTGTTTCCA iSNPI T F CCATCACACTATCCTGACATGAACAAAT iSNPI T F G A AG A lTTGC A TCCC AGTG AA AG C AC

166 iSN I T F GCACITCATAAAGAATCAGTCAGGATGC

167 iSNPI T F GGAGAATCAGGAAATAGTCAdTCCTAC

168 r.6811238 iSNPI T F CATTTGACCTTCTAGCCAAATGAAGTAC

169 rs?0411S8 iSNPI T F GGAATTTCTGAGAATAACATTGCCTCTC

170 iSN i T F CAT TGTTGGGGGAGCTAAACCTAATGA

171 rs719366 iSNP! T F CACTGTGACCACAGCATCTTTTAACTC

172 rs722098 iSNP! T F2 GGGTAAAG AAATATTCAGCACATCCAAA

173 rs722.2.90 iSNP! T F GAGTATCCCTTATCTAAAATGCTGGTCC

174 rs727811 iSNP! T F CTTTTTCTCTTACCGGAACTTCAACGAC

175 rs7291.72 iSNP! T F CCTCATTAATATGACCAAGGCTCCTCTG

176 rs733164_iSNP!J^"_F TGACTCTAATTGGGGATGTGGTAATTAG

177 rs73 155 iS?>iP! T F GACCTAACCFGGAGAAAACCGGAGA

178 rs iS?>iP! T F GTTTCrCTTCTCTGAACCTTTGTCTCAG

179 rs740910 iSNP! T F GCAAACACACAAAGATAGGTTCGAGTTT

180 rs763869 iSNP! T F CATATCAAGTGCTTTCTGTTGACATTTG

181 rs8037429 iSNPi T F CTG AAAAGTGCTACGTAAGAGGTCATTG

182 rs80 iSNPi T F CATCTGAGTGTGAGAAGAGCCTCAA

183 rs826472 iSNPI T F2 L C AG CAA A A AC MCI 11 IC! CtAG 1 AA 184 rs8731 iSNP! T F G(.1 AGGAAAG 1111 i 1 1 GG i.ACA

185 rs876724 iSNP! T F GAATATCTATGAGCAGGCAGTTAGCAG

186 rs891700 iSNP! T F2 CTAATCAGTGTCACl AFGTGVGAGCi^'AV

187 iSNP! T F CATCATACAGACTCAAGGAGCTTAGCTG

188 iSNP! T F CTTTCCAAGCCTTGGAAAACACAGAAAA

189 iSNP! T F GTACCTTATAAATCACGGAGTGCAGAC rs917118 iSNP! T F CAAGTGGTAAG AGATG ACTGAGGTCAA

191 rs93S233 iSNPI T F CTTCTTCTCTTAGAAGGACACTGGTCAG

192 rs964681 iSNPI T F GTTATGGAGGATTGGTAAGAACCAGAG

193 rs987640 iSNPI T F GAGCTGTTTAAGGGTAAAGGGGTAGTTA

194 iSNPI T F GCAGACAAAACCATGACAATGATCTTAG -S993934 iSNPI T F CCCATGATGAAACAGTTTGCACTAAATG iSNPI T F CICAAI 11 ICI I KX IGCI 1 ICAI

197 iSNPI S R2 ^'ITAGAAA^'ITCCAGATAGAGCTAAAACTG

198 iSNPI S R GTTAGGAAAAGAACCCAGGTGTTTT

199 iSNPI S R2 GCAAAAGTAAATACAAAGGCATACTTT

200 rsl028S28 iSNPI S R2 CAATGCAAAAGAAAGGTCCTTACTCGAC

201 iSNPI S R2 C Ai C TA A ACTCT A A A AC A A AC AT5TG

202 iSNPI S R2 GGTCCITAACCTATTAAATTTTAATGAG

203 rsl0488710 iSNPi S R. GA 111 CAA i i iAltii CAGCA 111 AAAA rsl0495407 iSNPi S R CCTCTTG GTTG C ATTG G ATTCTC ATTG

205 rsl058083 iSNPi S R TCTCCATGAAACTTGGGTTAATTTTGC

206 rs!0773760 iS P! S R TGTCTGGAAGTTCGTCAAATTGCAG

207 rs!294331 iS P! S R2 GTAGCATAAAACATTCCAAAAATTCAAT

208 rs!2997453 iSNPI S R TGCTITAAAGATACAGGITATCTGTATTAC rsl31S2883 iSNPI S R CICI C I IA I 1 I i K. IGCCI 11 rsl3218440 iSNPI S R GATCCTGAGATTCACCTCTAGTCCCT

211 rsl335873 iSNPi S R CCGTACCAGGTACCTAGCTATGTACT

212 rsl336071 iSNPi S R2 CnTCTGTrTTGTCCATCTGAAA^'ITCT

213 rs1355366 iSNPi S R CAAAGTTAAGTATCACCATCCAGCTGG

214 rs!357617 iSNPi S R ATAGGGATAGCTGATAAGAAACATGACC rsl3S2337 iSNPi S R CTTA ATA AG ACG CTG C ATCTGCCC A rsl413212 iSNPi S R TCC AGG AG AC ATTTGTTCATATAAGTG A

217 iSNPI S R AGA AC! ! ! !CA IA!CCAI 11 AG A A AC

218 iSNPI S R b! i !CACA! IGCAI C! !GG I rsl493232 iSNPI S R CCAAAGC 1 A ! ! 1 1 111 GG 1 C iSNPI S R G AAAG ITCACrr C AG ATGi C AAAGCC

221 rsl523537 iSNPI S R GGGTTTCAGTCr^'GCAACAAGATCTTG

222 rsl528460 iSNPI S R TGGAGATCAATATTTAGCCITAACATAT iSN i S R GACTGTTTCTCATCCTGTTATTATiTGT rsl736442 iSNPI S R AACACACAGAAACATCAAGCTGAGC iSNPI S R TTCCTGACATTCTCCTTCTTCTATCTG iSNPi S R ^■AI ACGCCI GAi i i i ACAACAAC

227 iSNPi S R CAGAGACTATGGATGGTATTTAGGTCAA

228 rs201.6276 iSNPI S R ACTTTGTGTGGCTGAGAGAGAGAAA rs2040411 iS P! S R TGAGTGTTCTCTGTATTTTCTTACTCTAAG rs2046361 iS P! S R2 A i i i i i GG ! CA■■ G 11 GACAL 11 tACC rs2056277 iSNPI S R2 GGTGTTAGGGAGACAGGCATGAATG rs2076848 iSNPI S R TGAAAcrrrrcAAcrcTCCTACCGCc rs2107612 iSNPI S R GTTAAAATTGCCACTAATTATGTGTTTT iSNPi S R A ACT G AT CC TATGC AG C A AG ATC TTTG iSNP! S R G AT G CTTG C A A AC A A AG ACT G A AAAG G

236 iSNP! S R GTCTGTGTGTCCT^'CTGAGATGATGAATG

237 rs2342747 iSNPi S R G GG AG G AAG AA AC AG AG AGTCiTG A

238 rs2399332 iSNPi S R AGTTTGTTGGCTTCTTTTGAGAAGTATC rs251.934 iSNP! S R GGCAGATGAAGTAGTAGATATCTGGCTG

240 rs279844 iSNP! S R GTTCAGTGTCAATTTTGACCAGATATT

241 rs2830795_iSNPI_S_R AGACATAGGACACACCATTTTATTGTCT

242 rs2831700 iSNPI S R TCAAAATATTTGGCTAAACTATTGCCGG

243 rs2920816 iSNPI S R2 CTGGAGTTATTAATAAATTGGATTATATAGC

244 rs321198 iSNP! S R TTACCTGTTTTCCTTTTGTGATTCCAC

245 iSNP! S R ACCAAGTCAAGAGCTCTGAGAGACAT

246 rs354439 iSNPI S R ACAGTGAATGATAT CAGAATATTGTGC

247 rs37S0962 iSNPi S R GAACAAGGTCAAGATATCAGCTTTCACC rs430046 iSNPI S R AGGTCATACAATGAATGGTGTGATGT

249 rs4364205_iSNPI_S_R ATCCACCCATGAGAAATATATCCACAA iSNPI S R ACAATTCAAATTAATGTAAAAACTGCAAGTG

251 iSNPi S R TAGTTCTAGTGTGGGATCTGACTCC

252 rs5606Sl iSN I S R GAACA ! ! G I I CAGG 1 1 I I C I CCA 1 C

253 -S6444724 iSNPI S R GAAAGGACTAAATTGTTGAACACTGGT

254 rs681123S iSNPI S R TGTGTGTTTTAAAGCCAGGTTTGTT iSNPI S R GATGGACTGGAACTGAGGATTTTCA iSN i S R AGCTTTAGAAAGGCATATCGTATTAACTG rs719366 iSNPi S R TTATAGTGAGTAAAGGACAGGCCCC iSNP! S R2 ACACATCTGTTGACAGTAATGAAATATCC rs722290 iSNP! S R GTTTAAACTTGGATACCATCCCCAAGAC

260 rs727811 iSNP! S R ATGAGATTGCTGGGAGATGCAGATG

261 rs7291.72 iSNP! S R CACATTTCCCTCTTGCGGTTACATAC

262 rs7331.64 iSNP! S R GACAAGCC 1 CG 1 1 GAG 1 1 1 1 i i i

263 rs735155 iSNP! S R TGTG AG AG TGTC ACCG A A 1TC A ACG

264 rs iSNP! S R AAATAGCAATGGCTCGTCTATGGTTAG

265 rs 740910 iSNP! S R TGCTAAGTAAGGTGAGTGGTATAATCA

266 rs763869 iSNP! S R ATAAATATGATGTGGCTACTCCCTCAT

267 rs8037429 iSNPi S R GCTACACCTCCATAGTAATAATGTAAGAG

268 rs80 8417 iSNPi S R TGAAGCAGCTAGAGAACTCTGTACGT

269 rs826472 iSNPI S R2 TTTTGTCTCTGTTATATTAGTCACCTATCTC

270 rs873196 iSNPI S R ATAGCCCTGCATTCAAATCCCAAGTG

271 rs876724 iSNPI S R TCCATTTTTATACCACTGCACTGAAG

272 rs891700_iSNPI_S_R2 G AG 1 AAA.AC .1 1 1 I A I CAAA l ! I CA

273 iSNPI S R TCTG G GTG C A A ACTAG CTG A ATATC AG

274 iSNPI S R GAAAATCTGGAGGCAATTCATGATGCC iSNPI S R ATACAATGATGATCACACGGGACCCT iSNPI S R CCATGAAGATGGAGTCAACATTTTACA

277 iSNPI S R TCCTAACCCCTAGTACGTTAGATGTG r.964681 iSNPi S R GAGGTG ATTTCTGTG AGG AACGTCG rs987640 iSNPI S R GTACATTCACTTAACAGGCTCTCTTTCC rs990S977 iSNPI S R AAITCATGAGCTGGTGTCCAAGGAG rs993934 iSNP! S R ATAACAGTCTCCAGAGTATATTAGCTTAG rs9951171 iSNPi S R GTTCCTCTGGGATGCAACATGAGAG rs!0497191 aSNPI T F GAAAGGATGAAGAGGGTGGATATTGGAG rs!079597 aSNPI T F CCAAACCTC CATCTCTTACCTGG ATT rsl 1652.805 aSNPI T F GTCCAAAGTCAAGTGCAAGTATAGTTGG rsl22.9984 aSNPI T F ACAATCrrrrCTG AATCTG AAC AGC TTC

287 rs!2439433 aSNPi 1^" F CAAAGGAAGGCATTTCCTAATGATCTTC

288 rs!2498138 aSNPi 1^" F CTTTGCTTTGCmTCTTCTTCAGGGAA

289 rsl2913832 _.pSNPLNU. T__F CTGCTTCAAGTGTATATAAACTCACAGT rs aSN I Ϊ F CCTAGGAAAGCAGTAACTAATTCAGGAG

291 rs aSN I Ϊ F GCAATTTGTTCACTTTTAGTTTCGTAGC

292 aSNPI T F GGCCTAATATGCATGTGTTCATGTCTCT

293 aSNPI T F C AG AGTTTCTC ATCTACG AA AG AGG AGT 294 rs aSNPI T F ATCCTAGACCTCCAGGTGGAATGATC

295 rsl7642714 aSNPI T F CTTGGCTGTCTCAATATTTTGGAGTAAG

296 rsl800414_aSNPIJ^"_F GAGTAAATGAGCTGrGGTTTCTCrCTTA

297 rsl834619 aSNPI T F CTTTCCATGTGGACCCITFAACATTCAG

293 aSNPI T F GCATAGTGAGCTGTTGATAGAGCTTTTG

299 rsl919550..aSNPI.T. F CTAGAACAAAATCATTGGCTCTCCTAGT

300 rsl92655..aSNPI.T. F GTCTGGTGAGTACTGGCTGAATGTAAA

301 rs200354_aSNPI_T_F CCAGAGGATGCTGCTAAACATTCTACAA

302 rs2024566 aSNPI T F GCTCATGCCTGGAATTCACCTTTATTTT

303 rs2042762 aSN PI T F CTAACTAGACATTTGGGCCACCTTACTT

304 re?.166624_aSNPI_T_F GTCTATGGTGCCTATAGAATGTACAGGT aSNPI T F CCCTCTCAAGTTTGTGAGCAAATATCAC

306 rs2238151 aSNPI T F CTCTATCTTG CTG C A ATG G ACTTTCC re260690_aSNPI_T_F CCVAGAAACAGA^'iTTTGAAGGGCiCTVG

308 rs231477S aSNPI T F AAATGAGGGGCATAGGGA AAGGGA

309 aSNPI T F CCTAGAAATCTGATACGTTATCCTATGA

310 rs3737S76. aSNPI. T..F AGGAGAGATATATTCAACATGAACCCAA

311 rs3811801. aSNPI. T..F GAACATCTCTGACCAGAAATTTCCAGTA

312 rs38231S9. aSNPI. T..F GTGTAGTG AAATCCTTAG ACTTAGGTAA

313 rs3916235_aSNPI_T_F AATACATGAAAAAGTAATACATGGGGCA

314 rs4471745 aSNPI T F ATTAAATGTTTACTTCTATCTACAAGGA

315 rs4833103 aSNPI T F CATTTTGTGAAATGCAAAGGGCAAATCT

316 rs4891.825 aSNPI NU T F GCTGAGAGGCTTAATTCCATCAAGATGA

317 rs491.8664 aSNPI NU T F CCCATCCTAAACTTAGTTTTATGGGCAG

313 rs6754311_aSNPIJ^"_F GTAACACATTCTCITTGGGAAGCTAGC

319 rs6990312 aSNPI NU T F CTTAGCTTCAGTGAAAATGG^'I CCTCTC

320 rs7226659 aSNPI NU T F CTTTC^'iTAGC iCCrCTCCAl^'l^'FCTCTTC

321 rs7326934..aSNPI. U..T .F GTCTATGCAGTGCTTCACTGAGGATTAT

322 aSi\!P! N U. T..F CTCTATCTGCTCAGAGCCTGCTTAAAAG

323 rs7554936..aSNPI. U..T .F GGAAAGGATACAGTGTTGAGCAAGATAG

324 rs7657799_aSNPI_NU_T_F GCCAACTTGATTCTCTTTCAAATGCTTG

325 rs7722456 aSNPI T F AGATGGGGTTTACCATGTTTCCCAG

326 rs798443 aSNPI T F GTACAGTAGTTAGTTTCCAGACTGATGA

327 re7997709_aSNPI_T_F GTAAATATCTAACTGTGTTTCCCTCAGT

328 aSNPI T F GAACCAAAAGGAATTAAGAGACTAGGGG

329 IS917115 aSNPI T F U I 1 1 l A GGC I I ! I CU 1 I ! i f.

330 aSNP! S R CCCACATCCTTCCCATTTATAGGCAA

331 aSN I S R TAC ATG ATCC 1^"A AGG GC AG C AG G A A

332 -S116S2805 aSNP! S R GTTTGGTGCATCCTCTTTCTCTCTC

333 rsl229984. aSNPI..S..R G ACTGT AGTCACCCCTTCTCCAACA

334 rsl2439433. aSNPLS .P- AGAGTGAAATACATAGAAAAGAAACTTAAAG

335 rsl2498138. aSNPLS .P- ATTTGCGAGAAACAGATAAATATTGAAG

336 rsl2913832_pSNPi_NU_S_R ACAGGAACAAAGAATTTGTTCTTCATGG

337 rsl426654 aSNPI S R CCTTGG ATTGTCTC AGG ATGTTG C A

338 rsl462906_aSNPI_S_R CTGGGATGTTTGTTTTGGCTTTGTG

339 rsl572018 aSNPI S R ATTGGTAGTACACTAATGGATATATGTGAG

340 rsl6891982 aSNPI S R GAATAAAGTGAGGAAAACACGGAGTTG

341 rsl74570_aSNP!_S_R GAGAGAGGCAGAAAGGAGGGATGAA

342 rsl7642714 aSNPi S R TACrcrGTCTTCAGTAGCrGTTTCrTGG

343 rsl800414 aSNPI S R TTAGACTCACCAAGATCAAGATGAATGC

344 rsl834619..aSNPLS. R ATCTC A ATA A AG CTG TTC A A A AC AG A A AG

345 rsl876482..aSNPLS. R TAAAGAAAATGCCATGGGCTGTACCC

346 rsl919550__aSNPI_.S_.R ATTGTGCAGCAGAACAGAGTGTAGTG

347 rs!92655 aSNPi S R ATTCTTTG C ATAG CTC ACG A A ATTTC CC

348 rs200354 aSNPi S R AAAATGAGACCTCGTATCTTTGCAGC 349 rs2024566 aSNPI S R AAATGCAGAACTGCCAAAAGAAACCC rs2042762 aSNPI S R GAGAATCTGTGAATGCCAGGGTCTG

351 rs2166624_aSNPI_S_R ATGGAITCATGITVCAGACATCTAAIT aSNPI S R ATCACTAGAAAGAAAAGAGTVCCTATTC

353 rs223S151 aSNPI S R G AAG TTTA AA AG AG 1^"G GG AAC ATG G GG

354 rs260690__aSNP! _ S._.R CTACGTAAGCAAAAATGATCACGCAC

355 rs2S14773__aSNPI_ S._.R AACCTGATGGCCCTCATTAGTCCTT

356 rs3i0644__aSNPi.__S._ R CACCAGATTTCTAGGAATAGCATGTGAG

357 rs3737576 aSNPI S R A AG AG CATAGTG AGG GGTTAG AC CT

358 rs3811301 aSNPI S R CTTTATATTTAGTGTAGAGATCAGTCTCC

359 rs3823159_aSNPI_S_R TGAGTCCTTTACCTAATCTTGGTTGTC

360 aSNPI S R AA 1 CAAAGCAA 1 1 L 1 1 1 1 ACCA

361 aSNPI S R TTTACTGGAACCCTGATTTTGTTGGA

362 rs4833103_aSNPI_S_R TGCCACTGATATA CAG VACCTGAGT

363 rs4891S25 aSNPI NU S R ACAAVCTCAAVCCCCCITAAT^'GTTTTC

364 rs4918664 aSNPI NU S R GTGGGCAG AG AGAG TAAG AGAACC1^"

36S rs67S4311_ aSNPI__S__R CAAACCAGATTCTGGCAGAATAGTTAGC

366 rs6990312. aSNPI..NU...S ..R C1TCTCTCCCATCCTCC1TCTCCAC

367 rs?2266S9_aSNPI_NU_.S_R AGATCAAGGGATCTGTGGGACAATAAC

368 rs7326934._ aSNPI._NU._S _R GGGGAGTGATTTCAAGCATCCTGATT

369 rs735480 aSNP! NU S R C ATG AGTTTG AG GTA AG ATG A AGG AG A

370 rs7554936 aSNPI NU S R TCTCTCTCATCCTAGTGAATGCCATC

371 rs7657799 aSNPI NU S R GGGTGATGATCTACCTTGCAGGTATA

372 rs7722456 aSNPI S R CTCAAGGCCCTGGGTCTGAAATTAC

373 rs79S443_aSNP!_S_R ACAVCTCCAGTTAATAAITVCCACVAAC

374 rs 997709 aSNPI S R TGGA^'ITGCTCAACAAATAGTGCTAAAA

375 rs870347 aSNPI S R CAT^'GCGACAVCCAGGTAGCVAAAATAC

376 rs917115__aSNP! _ S._.R ATGGATAAAAATGGAACTTTCAAGAGAA

377 rsl2203592 _pSNPi__T__F GTTTTATGTAAAGCTTCGTCATATGGCT

378 rsl2821256 _pSNPi__T__F GTTCCAACTTAGTCATAAAGTTCCCTGG

379 rsl2896399_pSNP!_T_F GGGTCTTGATGTTGTATTGATGAGGAAG

380 rsl393350_pSNP!_T_F CCTAACAGAAAGTCACTGTTTGTATCTG

381 rsl800407_pSNP!_T_F TCACTCTGGCTTGTACTCTCTCTGTG

382 rs2378249_pSNP!_T_F GGCTGGTTTCAGTCTGGAGACTTTATTT

383 rs2402130_pSNP!_T_F CTTCACCTCGATGACGATGATGATGAT

384 rs4959270_pSNP!_ T_F GACAATAACAGCACAAAGGATGGAAAAG

385 rsl805009_pSNP!_T_F GAACCAGACCACACAATATCACCAC

386 rs28777 pSNPi T F TCTACCFCnTGATGTCCCClTCGATAG

387 rsl68919S2_pSNP!_T_F CAGAGTTTCVCATCTACGAAAGAGGAGT

388 rs683..pSNPI. T..F CCCAGCTTTGAAAAGTATGCCTAGAACT

389 rsl2913832..pSNPLT. F CTGCTTCAAGTGTATATAAACTCACAGT

390 r5l2203S92__pSNP ._S._R TTGTTTCATCCACTTFGGTGGGTAAAAG

391 rsl2821256_pSNPi_S_R ^■AA I l AAGC I C I G i G ! ! ! AGGG I 1 ! ! !

392 rsl2896399_pSNPi_S_R CAATTCTTTGTTCTTTAGGTCAGTA AT

393 rs3393350_pSNPI_S_R TACTCTTCCTCAGTCCCTTCTCTGC

394 rsl800407_pSNPI_S_R TGAGACAGAGCATGATGATCATGGC

395 rs2378249_pSN PI_S_R GC AC A AGTCTAGG A ACTACTTTGCAC

396 rs2402130_pSNPI_S_R GAAGTATTTGAACCATACGGAGCCC

397 rs4959270_pSNPI_S_R TGAGGAACACATCCAAACFATGACAC

398 rsl805009_pSNPI_S_R TTTCTCGCCCT^'CATCAT^'CVGCAATG

399 rs28777__pSN PI __S_.R TCAGTTGATTTCATGTGATCCTCACAG

400 rsl6891982 _.pSNPI_.S__R GAATAAAGTGAGGAAAACACGGAGTTG

401 rs683__pSNP!._ S._ R ATTACCTTCTTTCTAATACAAGCATATG

402 rsl2913832_pSNP!_S_R ACAGGAACAAAGAATTTGTTCTTCATGG Example 2-DNA profiling for databanking

[0108] This example describes an experiment following the workflow of FIG. 2. This example does not utilize UMIs, as it, could be assumed that the samples obtained are from individuals whose identity is already known.

[0109] For this experiment, STRs are multiplexed with iSNPs as found in Table

.·> .

Table 3 -Identity informative SNPs and STRs

identity informative SN Ps

rsl005533 rsl357617 rs2076848 rs4530059 rs763869 rs 10092491 rsl360288 rs2107612 rs4606077 rs8037429 rsl015250 rsl382387 rs2111980 rs560681 rs8078417 rsl024116 rsl413212 rs214955 rs576261 rs826472 rsl028528 rsl454361 rs221956 rs6444724 rs873196 rsl029047 rsl463729 rs2269355 rs6811238 rs876724 rs!031825 rsl490413 rs2342747 rs6955448 rs891700 rsl0488710 rsl493232 rs2399332 rs7041158 rs901398 rsl0495407 rsl498553 rs251934 rs717302 rs907100 rs!058083 rsl523537 rs279844 rs719366 rs914165 rsl0773760 rsl528460 rs2830795 rs722098 rs917118 rs!0776839 rsl59606 rs2831700 rs722290 rs938283 rsll09037 rsl736442 rs2920816 rs727811 rs964681 rs!294331 rsl821380 rs321198 rs729172 rs987640 rsl2997453 rsl886510 rs338882 rs733164 rs9905977 rs!3182883 rsl979255 rs354439 rs735155 Γ5993934 rsl3218440 rs2016276 rs3780962 rs737681 rs9951171 rsl335873 rs2.040411 rs430046 rs740598

rsl336071 rs2046361 rs4364205 rs740910

rs!355366 rs2056277 rs445251 rs7520386

Autosomal STRs

D1S1656 CSF1PO vWA D21S11 D4S2408

D2S441 D7S820 D13S317 TPOX D17S1301

D2S1338 D8S1179 Penta E SE33 D9S1122

D3S1358 D10S1248 D16S539 Penta D D6S1043

FGA THOl D18S51 D22S1045 Amelogenin

D5S818 D12S391 D19S433 D20S482

X STRs

DXS8378 DXS8377 DXS10101 DXS10148 DXS10146

DXS7132 DXS10135 DXS10134 DXS10079 HPRTB DXS10074 DXS7423 DXS10103

Y STRs

DYS456 DYS393 DYS437 DYS533 DYS449

DYS389I/II DYS391 DYS438 DYS518 DYS522

DYS390 DYS439 DYS448 DYS570 DYS505

DYS458 DYS635 DYS576 DYS6 3 DYS627

DYS19 DYS392 DYS481 DYS460 DYF387Sla/b

DYS385a/b YGATAH4 DYS549 DYS612

[0110] Additional SNPs and STRs could of course be added to the above list. Examples of other potential targets include, hut, are not limited to, those markers found in Table 4.

Table 4-Examples of additional STRs and SNPs for multiplexing

[0111] Primers were designed to contain a gene-specific PGR. primer sequence at, the 3 ' end and an adapter tag sequence at the 5' end. in this experiment, the forward primers contain the tag sequence for the TruSeq Custom Amplicon i5 adapters and the reverse primers contain the tag sequence for the TruSeq Small RNA kit 17 adapters. The tags can be used as amplification primer sites as well as sequencing primer sites.

Adapter 5 tag sequence 5 'TACACGACGCTCTTCCGATCT3 ' (SEQ ID NO:4()3) Adapter i7 tag sequence 5 ' CTTGGCACCCG AG AATTCCA3 ' (SEQ ID NO:404) [0112] To balance the amplification between the STRs and the SNPs in the multiplex, primer design parameters were modified for the SNPs as described in Example 1. The original set of SNP primers designed using Alumina's Design Studio were classic PGR primers - short sequences with high melting temperatures and little to no secondary structure. Design Studio was used to design TruSeq Custom. Amplicon Probes and to create the reverse complement of the down-stream probe to make the reverse PGR primer. These primers, however, did not multiplex well and one bad primer could turn the assay from, good to bad (e.g., ail primer-dimer and no product) (FIG 4.) In an attempt to create better primers for multiplexing, Primer3 (shareware) was used that contains a mispriming library feature. It was discovered that the PrimerS designed primers performed even more poorly in the multiplex assay than the Design Studio primers. Surprisingly, data were being generated showing that the STR primers were multiplexing well. It was observed that the poorly designed primer pairs directed to STR. targets did not cause multiplex failures as the SNP primers did. The STR primers are long, AT-rich, and have low melting temperatures, contrary to what is known as a "good" primer.

[0113] The SNP primers were redesigned following the parameters of Example 1. The primers were mixed together for all of the targets. For this example, primer pairs for 56 STRs were mixed with primer pairs for 75 iSNPs, aSNPs, and phenotypic-in formative SNPs. Polymerase (Hot-start Phusion 0 in this example) was added to a mastermix of ail of the components required for PCR and the primers were added. The mix was pipetted into wells of a PCR plate, but the amplification could also be performed in tubes, etc. DNA was added to the plate as purified DNA in 15 microliter volume, however lysed extracts of blood or buccal samples from swabs or non-treated filter paper, or directly from blood or buccal samples on FTA Cards, etc. could also be used. For this experiment, purified control 2800M DNA at 1 ng and 100 pg was used. The reactions were subjected to PCR for a determined number of cycles (in the case of the example, 25cycles) following the protocol:

[0114] After cycling, the plates were removed from the thermal cycler. The reaction was brought to 50 microliters with polymerase (Kapa HiFi, Kapa Biosysterns), PGR mastermix containing all of the components required for PGR, and a pair of adapters (one i7 and one i5 adapter). A second round of PGR was performed for a determined number of cycles (10 cycles in the case of the example) to generate the sequencing libraries, following the protocol:

98 °C - 30 sec ;

98 °C -i 15 sec i

55 °C -i 30 sec ;

72 °C - : 1 min

72 x: - 5 min

; io °c -i hold

[0115] After cycling, the plate containing the completed libraries was removed from the thermal cycler. At this point, the samples can be pooled by volume and purified as a single sample using magnetic beads (SPR1) for example. The samples also can be purified individually. The pool or the individual libraries can be quantified by using a qPCR-based method, by using a. Fragment Analyzer or Bio Analyzer, or by using PicoGreen and a plate reader (as in the case of the example). A skilled artisan will know the myriad of options for library quantitation. If the libraries are purified individually, they can be normalized to 2 nM each concentration and pooled by volume.

[0116] The pools of purified libraries were denatured, diluted, clustered and sequenced on the MiSeq sequencing instrument with a 350-cyele sequencing run and the two index reads. After sequencing, the samples were demultiplexed according to the adapter sequences and analyzed through the Forensics Genomics pipeline (Iliuniina, Inc.). The STR reads were separated from the SNP reads and analyzed independently. The STRs were analyzed using the algorithm described in a previous patent application (PCT/US2053/30867, incorporated herein by reference in its entirety). The repeat number(s) and any sequence variations were reported along with the read numbers. The SNPs were analyzed using a manifest and the calls were reported along with the read numbers. The relative balance between alleles (Miri/Max%), balance between loci (%CY), error rates, and stutter rates were calculated for the STR loci. The results for the STRs in the initial databanking multiplex are shown in FIG. 5A-C, The balance (mean balance 80%), stutter (~3%) and error rates (less than 5%) meet design input requirements for the loci included in this example. The %CV (-142%) was calculated using all 56 loci. Even though the primers used show inter-locus balance, further primer optimization is anticipated to improve the inter-locus balance. The calls for the known loci match published results for 2800M The results for the SNPs are shown in FIG. 5D-E. The coverage, allele calls, stutter and other artifacts for the 56 STR loci in the large multiplex are shown in FIG. 6. These graphs mimic the electropherograms generated by CE technology. The bars are analogous to peaks for the defined allele (X axis) and the read counts (Y axis) are analogous to R.FU. The coverage for the SNPs was anywhere from 10-2500X depending on the SNP, however every SNP that was multiplexed was counted and provided accurate calls.

Example 3-DN A profj ling for criminal casework

[01.17] This example describes an experiment following the workflow of FIG. 3. This example incorporates UMIs into the primers, as it could be assumed that the samples obtained are from individuals whose identity is not already known.

[0118] For this experiment, the STRs were multiplexed with iSNPs, aSNPs and phenotypic-informative SNPs as found in Table 5. Table 5 -case work STRs and SNPs

rsl805005 rs2228479 rs4959270 rsl2913832

Ancestry informative SNPs

rsl0497191 rsl7642714 rs2238151 rs4471745 rs7554936 rsl079597 rsl800414 rs2593595 rs459920 rs7657799 rsll652805 E S1834619 rs260690 rs4833103 rs7722456 rsl229984 rsl871534 rs2814778 rs4891825 rs798443 rsl2439433 rs!876482 rs310644 rs4918664 rs7997709 rsl2498138 rsl919550 rs3737576 rs671 rs870347 rsl2913832 rsl92655 rs3811801 rs6754311 rs917115 rsl426654 rs200354 rs3814134 rs6990312 rs9522149 rs!462906 rs2024566 rs3823159 rs7226659

rsl572018 rs2042762 rs3827760 rs7251928

rsl6891982 rs2166624 rs3916235 rs7326934

rsl74570 rs2196051 rs4411548 rs735480

[0119] This example includes UMIs for the STR primers. For these examples, only the STR primers contain UMIs, however both STR and SNP primers could include UMIs if desired and that option is not excluded from practice. For this example however only the STR primers incorporate UMIs for demonstration purposes. Unique molecule identifiers were introduced during two cycles of PGR (FIG. 3). First, as for Example 2, the PGR primers contain a gene-specific PGR primer sequence at the 3' end and an adapter tag sequence at the 5' end, the same as for tag sequences used in Example 2 for i5 and i7 sequences. In this experiment, the UMIs are positioned between the gene-specific primer sequence and the tag sequence. In the case of this example, there were five randomized bases used for the UM1 on both the forward and reverse primers. The primers were mixed together for all of the targets. The primer mix comprised of 26 Autosomal STR primer pairs and 86 SNP primer pairs (92 SNPs covered). Polymerase (Hot-start Phusion II in this example) was added to a. mastemiix of all of the components required for PGR, and the primers were added. The mix was pipetted into wells of a PGR plate. DNA was added to the plate as purified DNA, optimally 1 ng. As in Example 2, purified DNA from 2800M control was tested at 1 ng. The multiplex reaction mixture was subjected to two cycles of PGR following the protocol: ; 98C 3 min

98C 2 min

i 54C 12 min i ramp 0.2C/sec

72C 4 min ; 2 cycles i

4C. hold

[0120] After cycling, the samples were removed from the thermal cycler and E. coli single-stranded DNA binding protein (SSB) was added to the reaction. It was contemplated that the SSB reduces primer dimers by the unused tagged gene-specific primers and prevents any more amplification from these primers. The SSB was incubated with the sample on ice, alternatively RT or 37C incubation could also be used. After this incubation, polymerase (Hot-start Phusion II in this example) was added to a mastermix of all of the components required for PCR, and the mastermix was added to the sample with a pair of adapters (i7 and i5 adapters) and cycled for a determined number of cycles (in this experiment 34 cycles), following the protocol:

95C : 3 mi n

95C i 30 sec

66C i 30 sec

72C i l mi n

72C i 5 mi n

IOC i hoid

[0121] The samples were purified with SPRI beads, and the individual libraries could be quantified by using a qPCR-based method, by using a Fragment Analyzer (as in the case of the example) or BioAnalyzer, or by using PicoGreen and a plate reader. The libraries were normalized to 2 nM each concentration and pooled by volume,

[0122] The pools of purified libraries were denatured, diluted, clustered and sequenced using the MiSeq with a 350xl00-cycle sequencing run and the two index reads. After sequencing, data was determined as reported in Example 2. However, since the primers contain IJMIs, the UMls were used to collapse the data by using PCR duplicates to remove sequencing and PCR errors and artifacts. The SNPs were analyzed using a manifest and the calls were reported along with the read numbers. The relative balance between alleles (Min/Max%), balance between loci (%CV), error rates, and stutter rates (STRs, only) were calculated. The results for the initial casework multiplex are shown in FIG. 7A-E. The coverage, allele calls, stutter and other artifacts for the 26 STR loci in the large multiplex are shown in FIG. 8, These graphs mimic the electropherograms generated by CE. The bars are analogous to peaks and the read counts are analogous to RFU. The coverage for the SNPs was anywhere from 10-5500X depending on the SNP, however every SNP that was multiplexed was counted and provided useful results.

[0123] One result that was generated by these studies was that stutter was shown to be a PGR artifact. This has been hypothesized by many investigators (and polymerase slippage has been indicated in human colon cancers), but this hasn't been demonstrated for the Forensics assays. The UMIs can be used to show that, stutter is indeed a PCR artifact. The products with n+1 or n-1 repeats have the same UMIs as the products with the correct number of repeats (FIG. 9). In FIG. 9A each locus shows the results without UMI correction compared to FIG. 9B where UMI correction is performed. As demonstrated, without UMI correction the balance between the alleles is not, as good as when UMI correction is performed. Further, there is considerably more stutter that is apparent without UMI correction. The portion of the bar above the inter-bar line represents the sequencing error whereas below the line represents the correct sequence within the STR sequence. The error is greatly reduced with UMI correction. For example, SE33 locus has error that is removed with UMI correction. Error correction can be extremely important for criminal casework to provide the most accurate DNA profiling possible.

Example 4-DNA profiling using 12 sample individuals

Methods and Materials

[0124] DNA from 12 sample individuals (Sample #: 1, 3, 4, 5, 6, 7, 10, 13, 14, 15, 16, 17) and one reference genome (2800M) was tested following the workflow of FIG. 3. This experiment incorporates UMIs into the STR primers as described in Example 3. Two replicates of each sample were analyzed with the Illurnina® ForenSeq DNA Signature Library Prep Kit on the MiSeq sequencer. One ng DNA was used for each replication using the DNA Primer Mix B: Collected samples mix, which contains primers for 61 STRs plus Amelogenin, 95 identity-informative SNPs, 56 ancestry-informative SNPs, 22 phenotypic- informative SNPs (2 ancestry SNPs are also used for phenotype prediction). Default Settings

[0125] STR: analytical threshold = 6.5%; interpretation threshold =15%. SNP: analytical threshold = 3%; interpretation threshold = 15%.

[0126] The high level sequencing calls, such as coverage and loci called, for the DNA profiling of the 12 sample individuals are shown in FIG. 12. As can be seen, every locus was covered by at least 100,000 reads in both replications. Only two samples resulted in a failed STR call (1 out of 61). All of the 173 SNPs were successfully called in all individuals in both replications. Sample STR calls of two sample individuals are shown in FIG. 16. Sample SNP calls of two sample individuals are shown in FIG. 17. FIG. 13 shows the population statistics, such as the random match probability (RMP) of the National Institute of Standards and Technology (NIST) auto-STRs, 95% confidence haplotype frequency of NIST Y-STRs, RMP of the dbSNP iSNPs, and the RMP of the STRs from the U.S. Y-STR database.

[0127] Phenotypes, such as eye color and hair color of the 12 sample individuals and the reference individual were predicted based on the genotype of pSNPs in the experiment, and compared to the self-reported phenotypes (FIG, 14), High degree of correlation between the predicted and reported phenotypes was observed.

[0128] Ancestry of the 12 sample individuals were predicted using the genotype of 56 aSNPs in the experiment. PCA1 and PCA3 scores of each sample individual were calculated, and plotted against reference samples on an ancestry plot. As shown in FIG. 15, ancestry of the sample individuals can be predicted based on the location on the ancestry plot. Fourteen centroid points were included in the ancestry plot (circles). Based on the closest centroid point, the ancestry of each sample individual was predicted.

[0129] The DNA profiling experiment also showed high level of intra-locus balance in both the STR loci and the SNP loci, as can be seen in FIG. 18, and low level of stutter, as can be seen in FIG, 19.

[0130] Six of the 12 individuals plus 2800M have at least one isometric heterozygote locus, which is shown in FIG. 20. An isometric heterozygote locus is defined as an STR that has the same repeat number, two different sequences, which are equally balanced. Using the information on the variants in the STR D8S1179, the 13 allele of Sample 15 was traced to the grandmother, Sample 17 (FIG. 21). Similar variant information in the STR D13S317 was used to trace the alleles of Sample 15. However, in this case the origin of either allele cannot be ascertained (FIG. 22).

Example 5-DNA profiling for research, forensic, or paternity use

[0131] This example is based on the workflow described in ForenSeq™ DNA Signature Prep Guide (Alumina, San Diego, CA), the content of which is hereby incorporated by reference in its entirety.

[0132] Either purified DNA or crude lysate may be used for this example. For purified DNA, each 1 ng sample is diluted to 0.2 ng/μ] with nuclease-free water. For crude lysate, each 2 μΐ sample is diluted with 3 μΐ nuclease-free water. A Master Mix is set up for eight or more reactions. For each reaction, 5.4 μ,Ι of ForenSeq PCR1 Reaction Mix, 0.4 μΐ of ForenSeq Enzyme Mix and 5.8 μΐ of DNA primer Mix (A or B) are added into a 1.5 ml microcentrifuge tube. 10 μΐ of Master Mix is transferred to each well of the PGR plate, and the DNA or lysate is added. The multiplex reaction mixture is subjected PGR following the protocol:

98 degrees C for 3 min.

8 cycles of:

96 degrees C for 45 sec.

80 degrees C for 30 sec.

54 degrees C for 2 min., with specified ramping mode

68 degrees C for 2 min., with specified ramping mode

10 cycles of:

96 degrees C for 30 sec.

68 degrees C for 3 min., with specified ramping mode

68 degrees C for 10 min.

Hold at 10 degrees C.

[0133] After cycling, the samples are removed from the thermal cycler. ForenSeq PCR2 Reaction Mix is added to the samples with a pair of adapters (i7 and i5 adapters) and cycled for a 15 cycles, following the protocol: 98 degrees C for 30 sec.

15 cycles of:

98 degrees C for 20 sec.

66 degrees C for 30 sec.

68 degrees C for 90 sec.

68 degrees C for 10 min.

Hold at 10 degrees C.

[0134] The samples are purified with Sample Purification Beads, and the libraries are normalized and pooled by volume. The pooled libraries are diluted in Hybridization Buffer (HT1), added with Human Sequencing Control (HSC), and heat denatured in preparation for sequencing.

Example 6-Genotvpmg with degraded DMA

[0135] FIG. 23 shows genotyping results using sheared and/or DNase-treated DNA representing degraded DNA. As shown, more than 50% STR and SNP loci were correctly called with sheared DNA. A random match probability (RMP) of 10^"19 was achieved with DNA of less than 100 bp. Correct ancestry was also predicted using degraded DNA.

Example 7-Geno typing sensitivity

[0136] FIGS. 24 and 25 show genotyping sensitivity results at sub-nanogram DNA input levels from 7.82 pg to 1 ng. As shown, 100% alleles were successfully called at 125 ng input DNA for both STR and SNP. More than 50% alleles were successfully called at as low as 7.82 pg input DNA. Intra-locus balance was greater than 70% for most loci at 1 ng input DNA.

[0137] All numbers expressing quantities of ingredients, reaction conditions, and the like used in the specification are to be understood as being modified in all instances by the term "about". Accordingly, unless indicated to the contrary, the numerical parameters set forth therein are approximations that may vary depending upon the desired properties sought to be obtained. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of any claims in any application claiming priority to the present application, each numerical parameter should be construed in light of the number of significant digits and ordinary rounding approaches.

[0138] All references cited herein, including but not limited to published and unpublished applications, patents, and literature references, are incorporated herein by reference in their entirety and are hereby made a part of this specification. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and/or take precedence over any such contradictory material.

[Θ139] Citation of the above publications or documents is not intended as an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents.

[0140] Although the present invention has been fully described in connection with embodiments thereof with reference to the accompanying drawings, it is to be noted that various changes and modifications will become apparent to those skilled in the art. Such changes and modifications are to be understood as being included within the scope of the present invention. The various embodiments of the invention should be understood that they have been presented by way of example only, and not by way of limitation. Likewise, the various diagrams may depict an example architectural or other configuration for the invention, which is done to aid in understanding the features and functionality that can be included in the invention. The invention is not restricted to the illustrated example architectures or configurations, but can be implemented using a variety of alternative architectures and configurations. Additionally, although the invention is described above in terms of various exemplary embodiments and implementations, it should be understood that the various features and functionality described in one or more of the individual embodiments are not limited in their applicability to the particular embodiment with which they are described. They instead can, be applied, alone or in some combination, to one or more of the other embodiments of the invention, whether or not such embodiments are described, and whether or not such features are presented as being a part of a described embodiment. Thus the breadth and scope of the invention should not be limited by any of the above-described exemplary embodiments.

[0141] Terms and phrases used in this document, and embodiments thereof, unless otherwise expressly stated, should be construed as open ended as opposed to limiting. As examples of the foregoing: the term "including" should be read as meaning "including, without limitation" or the like; the term "example" is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof; and adjectives such as "conventional," "traditional," "normal," "standard," "known", and terms of similar meaning, should not be construed as limiting the item described to a given time period, or to an item available as of a given time. But instead these terms should be read to encompass conventional, traditional, normal , or standard technologies that may be available, known now, or at any time in the future. Likewise, a group of items linked with the conjunction "and" should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as "and/or" unless apparent from the context or expressly stated otherwise. Similarly, a group of items linked with the conjunction "or" should not be read as requiring mutual exclusivity among that group, but rather should also be read as "and/or" unless it is apparent from the context or expressly stated otherwise. Furthermore, although items, elements or components of the invention may be described or claimed in the singular, the plural is contemplated to be within the scope thereof unless limitation to the singular is explicitly stated. For example, "at least one" may refer to a single or plural and is not limited to either. The presence of broadening words and phrases such as "one or more," "at least," "but not limited to", or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent.

Claims (86)

WHAT IS CLAIMED IS:

1. A method for constructing a DNA profile comprising:

providing a nucleic acid sample,

amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat in a. multiplex reaction to generate amplification products, and

determining the genotypes of the at least one SNP and the at least one tandem repeat in the amplification products, thereby constructing the DNA profile of the nucleic acid sample,

2. The method of claim i , comprising generating a nucleic acid library from the amplification products.

3. The method of claim 2, comprising determining the sequences of the nucleic acid library.

4. The method of any one of claims 1-3, wherein the nucleic acid sample is from a human.

5. The method of any one of claims 1 -3, wherein the nucleic acid sample is from an environmental sample, a plant, a non-human animal, a bacterium, archaea, a fungus, or a virus.

6. The method of any one of claims 1-5, wherein the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the nucleic acid sample.

7. The method of any one of claims 1-6, wherein the DNA profile is used for one or more of disease diagnostics or prognosis, cancer biomarker identification, genetic anomaly identification or genetic diversity analysis.

8. The method of any one of claims 1-6, wherein the DNA. profile is used for one or more of databanking, forensics, criminal case work, paternity or personal identification.

9. The method of any one of claims 1-8, wherein each of the plurality of primers has a low melting temperature and'or has a length of at least 24 nucleotides.

10. The method of claim 9, wherein each of the plurality of primers has a melting temperature that is less than 60 degrees C.

1 1 . The method of claim 9, wherein each of the plurality of primers has a melting temperature that is about 50 degrees C to about 60 degrees C.

12. The method of any one of claims 9-1 1 , wherein each of the plurality of primers has a length of at least 24 nucleotides.

13. The method of any one of claims 9-1 1 , wherein each of the plurality of primers has a length of about 24 nucleotides to about 38 nucleotides.

14. The method of any one of claims 9-1 3, wherein each of the plurality of primers comprises a homopoiymer nucleotide sequence.

15. The method of any one of claims 5 -54, wherein the nucleic acid sample is amplified by polymerase chain reaction (PCR).

16. The method of claim 55, wherein the nucleic acid sample is amplified in an amplification buffer having a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers.

17. The method of claim 16, wherein the salt comprises KC1, LiCl, aCl, or a combination thereof.

1 8. The method of claim 16, wherein the salt comprises KC1.

19. The method of claim 18, wherein the concentration of KG in the amplification buffer is about 100 rnM to about 200 mM.

20. The method of claim 18, wherein the concentration of KCl in the amplification buffer is less than about, 150 mM.

21. The method of claim 18, wherein the concentration of KCl in the amplification buffer is about 145 mM.

22. The method of any one of claims 1-21 , wherein the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof.

23. The method of any one of claims 1 -22, wherein the plurality of primers specifically hybridize to at least 30 SNPs.

24. The method of any one of claims 1 -22, wherein the plurality of primers specifically hybridize to at least, 50 SNPs.

25. The method of any one of claims 1 -24, wherein the tandem repeat is a short tandem repeats (STR), an intermediate tandem repeat (ITR), or a. variant thereof,

26. The method of any one of claims 1 -25, wherein the plurality of primers specifically hybridize to at least 24 tandem repeat sequences.

27. The method of any one of claims 1 -25, wherein the plurality of primers specifically hybridize to at least 60 tandem repeat sequences.

28. The method of any one of claims 1 -27, wherem the nucleic acid sample comprises about 100 pg to about 100 ng DNA.

29. The method of any one of claims 1-27, wherein the nucleic acid sample comprises about 10 pg to about 100 pg DNA.

30. The method of any one of claims 1 -27, wherein the nucleic acid sample comprises about 5 pg to about 10 pg DNA.

31 . The method of any one of claims 1-30, wherein the nucleic acid sample comprises genomic DNA.

32. The method of claim. 31 , wherein the genomic DNA. is from a forensic sample.

33. The method of claim 31 or 32, wherein the genomic DNA comprises degraded

DMA.

34. The method of any one of claims 1 -33, wherein at least 50% of the genotypes of the at least one SNP and at least one tandem repeat are determined.

35. The method of any one of claims 1-33, wherein at least 80% of the genotypes of the at, least one SNP and at least one tandem repeat are determined.

36. The method of any one of claims 1 -33, wherein at least 90% of the genotypes of the at least one SNP and at least one tandem repeat are determined.

37. The method of any one of claims 1-33, wherein at least 95% of the genotypes of the at, least one SNP and at least one tandem repeat are determined.

38. The method of any one of claims 1-37, wherein each of the plurality of primers comprises one or more tag sequences.

39. The method of claim 38, wherein the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof.

40. The method of claim 38, wherein the one or more tag sequences comprise a primer tag,

41. The method of claim 38, wherein the one or more tag sequences comprise a unique molecular identifier tag.

42. A method of constructing a nucleic acid library comprising:

providing a nucleic acid sample, and

amplifying the nucleic acid sample with a plurality of primers that specifically hybridize to at least one target sequence comprising a single nucleotide polymorphism (SNP) and at least one target sequence comprising a tandem repeat sequence in a multiplex reaction to generate amplification products.

43. The method of claim 42, wherein the nucleic acid sample is not fragmented prior to the amplification.

44. The method of claim 42 or 43, wherein the target sequences are not enriched prior to the amplification.

45. The method of any one of claims 42-44, wherein the at least one SNP indicates the ancestry or a phenotypie characteristic of the source of the nucleic acid sample.

46. The method of any one of claims 42-45, wherein each of the plurality of primers comprises one or more tag sequences.

47. The method of claim 46, wherein the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, or a unique molecular identifier tag, or a combination thereof.

48. The method of any one of claims 42-47, comprising amplifying the amplification products with a second plurality of primers.

49. The method of claim 48, wherein each of the second plurality of primers comprises a portion corresponding to the primer tag of the plurality of primers and one or more tag sequences.

50. The method of claim 49, wherein the one or more tag sequences of the second plurality of primers comprise a capture tag, or a sequencing tag, or a combination thereof.

51 . The method of any one of claims 48-50, comprising adding single stranded- binding protein (SSB) to the amplification products.

52. The method of any one of claims 42-55 , wherein the nucleic acid sample and/or the amplification products are amplified by polymerase chain reaction (PGR).

53. The method of claim 52, wherein the nucleic acid sample and/or the amplification products are amplified in an amplificatio buffer having a salt conce tration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventio ally designed primers.

54. The method of claim 53, wherein the salt comprises Cl, LiCl, NaCl, or a combination thereof.

55. The method of claim 53, wherein the salt comprises KCl.

56. The method of claim 55, wherein the concentration of KCl in the amplification buffer is about 100 rnM to about 200 mM.

57. The method of claim 55, wherein the concentration of KCl i the amplification buffer is less than about 150 mM.

58. The method of claim 55, wherein the concentration of KCl in the amplification buffer is about 145 mM.

59. A nucleic acid library constructed using the method of any one of claims 42-

58.

60. A nucleic acid library comprising a plurality of nucleic acid molecules, wherein the plurality of nucleic acid molecules comprise at least one tandem repeat sequence flanked by a first pair of fag sequences and at least one single nucleotide polymorphism (SNP) sequence flanked by a second pair of tag sequences.

61. The method of claim 60, wherein the at least one SNP indicates the ancestry or a phenotypic characteristic of the source of the plurality of nucleic acid molecules.

62. A plurality of primers that specifically hybridize to at least one short target sequence and at least one long target sequence in a nucleic acid sample, wherein amplifying the nucleic acid sample using the plurality of primers in a single multiplex reaction results in at, least one short amplification product and at least one long amplification product, wherein each of the plurality of primers comprises one or more tag sequences.

63. The plurality of primers of claim 62, wherein the short target sequence comprises a single nucleotide polymorphism (SNP) and the long target sequence comprises a tandem repeat.

64. The plurality of primers of claim 62 or 63, wherein the one or more tag sequences comprise a primer tag, a capture tag, a sequencing tag, a unique molecular identifier tag, or a combination thereof,

65. The plurality of primers of any one of claims 62-64, wherein each of the plurality of primers has a low melting temperature and/or has a length of at least 24 nucleotides.

66. The plurality of primers of claim 65, wherein each of the plurality of primers has a melting temperature that is less than 60 degrees C.

67. The plurality of primers of claim 65, wherein each of the plurality of primers has a melting temperature that is about 50 degrees C to about 60 degrees C.

68. The plurality of primers of any one of claims 65-67, wherein each of the plurality of primers has a length of at least 24 nucleotides.

69. The plurality of primers of any one of claims 65-67, wherein each of the plurality of primers has a length of about 24 nucleotides to about 38 nucleotides.

70. The plurality of primers of any one of claims 65-69, wherein each of the plurality of primers comprises a homopolymer nucleotide sequence.

71. The plurality of primers of any one of claims 62-70, wherein the nucleic acid sample is amplified by polymerase chain reaction (PCR).

72. The plurality of primers of any one of claims 63-71, wherein the SNP is an ancestry SNP, a phenotypic SNP, an identity SNP, or a combination thereof.

73. The plurality of primers of any one of claims 62-72, wherein the plurality of primers specifically hybridize to at least 30 SNPs.

74. The plurality of primers of any one of claims 62-72, wherein the plurality of primers specifically hybridize to at, least 50 SNPs.

75. The plurality of primers of any one of claims 63-74, wherein the tandem repeat is a short tandem repeats (STR), an intermediate tandem repeat (ITR), or a variant thereof.

76. The plurality of primers of any one of claims 62-75, wherein the plurality of primers specifically hybridize to at least 24 tandem repeat sequences.

77. The plurality of primers of any one of claims 62-75, wherein the plurality of primers specifically hybridize to at least 60 tandem repeat sequences.

78. A kit comprising at least one container means, wherein the at least one container means comprises a plurality of primers of any one of claims 62-77,

79. The kit of claim 78, further comprising a reagent for an amplification reaction.

80. The kit of claim 79, wherein the reagent is an amplification buffer for polymerase chain reaction (PGR),

81. The kit of claim 80, wherein the amplification buffer comprises a salt concentration that is increased compared to the salt concentration of an amplification buffer used in conjunction with conventionally designed primers.

82. The kit of claim 81 , wherein the salt comprises KCl, Li CI, NaCl, or a combination thereof,

83. The kit of claim 81, wherein the salt comprises KCl.

84. The kit of claim 83, wherein the concentration of KCl in the amplification buffer is about 00 mM to about 200 mM.

85. The kit of claim 83, wherein the concentration of KCl in the amplification buffer is less than about 150 mM.

86. The kit of claim 83, wherein the concentration of KCl in the amplification buffer is about 45 mM.